THE EXPRESSION OF NESTIN IN THE INDUCED DIFFERENTIATION INTO NEURONS OF RAT BONE MARROW MESENCHYMAL STEM CELLS BY NEUROTROPHIN-3 (NT-3)

Objective: The aim of this study was to examine the role of NT-3 as a single neurotrophic factor in the expression of nestin in the neural differentiation of MSCs. Methods: MSCs were isolated from rat bone marrow and induced with NT-3 at concentrations of 20, 25, and 30 ng/ml for 7 and 14 d (the control was no NT-3). Nestin underwent immunocytochemical analysis on days 7 and 14. Five high-power random fields were documented. Results: A post-hoc analysis using LSD after one-way ANOVA test yielded a statistically significant difference in the percentage of nestin-positive cells in MSCs with NT-3 at concentrations of 20, 25, and 30 ng/ml for 7 d compared to the control group (p<0.05). The percentages of nestin-positive cells at concentrations of 20, 25, and 30 ng/ml, and in the control data on day 7 were 14.55±1.26%, 16.20±1.07%, 13.78±1.19%, and 9.81±0.79%, respectively. NT-3 at 25 ng/ml induced the highest MSCs neural differentiation on day 7 and remained constant until day 14. Conclusion: NT-3 plays a role in the early stage of differentiating MSCs from rat bone marrow into neurons, with the optimal concentration being 25 ng/ml

THE ROLE OF ACALYPHA INDICA LINN. EXTRACT ON HEART RATES WITH MYASTHENIA GRAVIS RAT MODEL

  Objective: Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ) caused by antibodies that attack components of the postsynaptic membrane, impair neuromuscular transmission, and lead to weakness and fatigue of skeletal muscle. Acetylcholine is also used as a neurotransmitter in the autonomic nervous system. Striated cardiac muscle can be a target for immune attack manifesting as heart failure, arrhythmia, and sudden death. Involvement of the heart rate (HR) has been claimed and reported, but a causal connection between MG and altered cardiac function has not been found.Methods: For this study of experimental autoimmune MG (EAMG) is used rocuronium, prostigmine, and Acalypha indica (AI) Linn. compared with HR.Results: From the results, the study found that sympathetic activity of HR variability in EAMG injected with rocuronium 10 mg/kg body weight (BW) in 10 min significantly found increasing in measures of short-term variations in HR variability, indicating parasympathetic impairment.Conclusion: We conclude that in MG, cholinergic transmission is affected more diffusely than previously thought. Furthermore, AI was given orally 30 mg/kg BW has an effect similar to the injecting of prostigmine 10 mg/kg BW that can reduce HR. Driven by the fact that the pharmacological treatment of MG is unsatisfied, it needs the therapeutic development for MG using herbal ingredients of AI. This means that the AI compositions containing anti-MG whose composition should be investigated for the next research

Isolation and Characterization of Adipose-Derived Mesenchymal Stem Cell Exosomes: an In-Vitro Study

Exosomes from Mesenchymal Stem Cells (MSC) is currently one of the highlighted research due to its more specific action on the target tissue. Exosome is one of the MSC Extracellular Vesicle (EVs) that acts as a mediator in the cellular communication. However, not so many literatures have succeed in elaborating its isolation and characterization process. This study aimed to explain the method used in exosomes isolation from Adipose-Derived MSC (ASC) and elaborate its characteristics. This is an in-vitro study performed in the Stem Cell and Tissue Engineering (SCTE) Indonesia Medical Education and Research Institute (IMERI) laboratory. Ultracentrifugation method was performed to isolate exosome from ASC. The exosome were characterized based on its particle size, morphology, and CD63 and CD81 expression for its purity. We were able to isolate sterile exosome from ASC by performing differential ultracentrifugation. The mean size of exosome was 88.7 nm ± 40 nm SD and showed expression of CD63 and CD81. Exosome was successfully isolated from ASC using ultracentrifugation method and characterization following the MISEV standard should be implemented in order to meet the therapeutic efficacy and safety issues as a regenerative agent

Effects of fasting on FOXO3 expression as an anti-aging biomarker in the liver

Background: Aging is a multifactorial degenerative process that can be modulated by fasting through activation of the Fork-head transcription factor of the O class 3 (FOXO3), which plays an important role in increasing lifespans. However, the effects of different fasting durations on the expression of FOXO3 in the liver has not yet been reported. Objective: This study analyzed the effects of different fasting durations on the FOXO3 expression and its pathway by measuring sirtuin1 (SIRT1), insulin-like growth factor-1 (IGF-1), and superoxide dismutase (SOD) activity in the liver. Methods: New Zealand white rabbits were used to mimic the effects of fasting on humans. The rabbits were divided into the control, intermittent fasting (IF), and prolonged fasting (PF) groups. Both fasting groups were interspersed with the non-fasting phase for 8 h. This treatment was conducted for 6 days. On Day 7, all the rabbits were sacrificed, and their livers were taken to measure the FOXO3 and SIRT1 mRNA expressions, the IGF-1 protein level, and the SOD activity level. ANOVA, multiple comparison, and Pearson's correlation were performed for statistical analysis. Results: The FOXO3 and SIRT1 mRNA expressions were significantly higher in the IF group than in the control group. The FOXO3 expression was also 2.5 times higher in the IF group than in the PF group. There was a positive correlation between the FOXO3 and SIRT1 mRNA expressions. The IGF-1 protein level was significantly lower in the IF and PF groups than in the control group. The SOD-specific activity level was significantly higher in the IF group than in the control and PF groups. Conclusions: Intermittent fasting significantly increased the FOXO3 and SIRT1 mRNA expressions and the SOD activity level in the livers of the rabbits and significantly decreased the circulating and hepatic IGF-1. Therefore, intermittent fasting may give a protective intervention effect towards aging

The Use of Cell-penetrating Peptide for Delivery of Recombinant Transcription Factor DNA into Primary Human Fibroblast

Background: Reprogrammed cell therapy has not been applied for clinical purposes due to the malignancy issue. The aim of this study was to design the recombinant vector of the transcription factors and analyze the effectiveness of cell-penetrating peptide delivering system for human primary fibroblast transfection to avoid the malignancy issue.Materials and methods: The constructions of CCAT/enhancer binding protein alpha (CEBPA), hepatocyte nuclear factor 4 alpha (HNF4A), nuclear receptor subfamily 1 group I member 2 (NR1I2) were confirmed with DNA digestion and sequencing. Breast reduction (BRED) and palate (PAL) tissue were used as human primary fibroblast sources. The transcription factors were delivered into BRED and PAL with recombination of avian leukosis sarcoma virus (ALSV), human immunodeficiency virus (HIV) matrix, and regulator of expression of virion proteins (Rev) (ALMR), tagged with enhanced green fluorescence protein (eGFP). Post-transfection cells were then cultivated with optimized medium. Gene expression was measured with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).Results: Gene expression levels of CEBPA, HNF4A, NR1I2, glutamate-ammonia ligase (GLUL), albumin (ALB), and cytochrome P450 (CYP) were increased. Transfection with ALMR, which were more efficient in BRED than PAL fibroblasts may have the advantage in autologous cell therapy for elderly patients.Conclusion: Transfection of transcription factors to human primary fibroblast may be performed by using constructions of plasmid as designed in this study.Keywords: recombinant plasmid, hepatocyte-like cells, primary fibroblasts, recombinant peptide, cell reprogramming, autologous cells therap

Comparison of 2D and 3D Co-Culture with Hanging Drop Method to Produce a More Clinically Relevant Microenvironment

2-dimensional (2D) co-cultures show limited resemblence with in vivo microenvironment while 3-dimensional (3D) co-cultures form a micromass which is more similar with in vivo microenvironmentthus it would be more useful in biomedical research. This study was intended to compare 2D co-cultures with 3D co-cultures of stem cells derived from umbilical cord blood vessels and hepatic stellate cells, conducted with hanging drop method to assess the cell morphology and the formation of spheroid from the micromass.. This in vitrostudy was conducted at Institute of Human Virology and Cancer Pathobiology (IHVCB) UI and histology laboratory from September 2015 to October  2016 using stem cells which were isolated from human umbilical cord blood (UCB) and LX-2 cell line (human hepatic stelate cells). Human umbilical cord blood was sorted with MACS CD34 and percentage of CD34+ cells were analyzed by flowcytometry. Stem cell co-cultures (UCB) or umbilical cord and L2 was did by hanging drop methods for 2D co-culture and  3D co-culture. Triplicates experiments were performed for each set of co-culture. The results showed the difference in the morphology of 2D co-culture and 3D hanging drop compared to monoculture. In the 2D co-culture there was a micromass formation, whereas    in the 2D monoculture, the micromass was not formed. In the 3D hanging drop there was a smaller spheroid compared to the 3D hanging drop monoculture. The morphology of 2D and 3D co-culture cells with hanging drop method in comparison with monoculture cells showed phenotypic changes of cells which were incorporated together in the micromass

Comparison of Maturation Stages of Natural Killer Cell Differentiation Culture from Cultured and Freshly Isolated Umbilical Cord Blood Hematopoietic Stem Cells

Background: Natural killer (NK) cells originate from the differentiation of hematopoietic stem cells (HSCs) in the common lymphoid progenitor pathway, and HSCs can be obtained from umbilical cord blood (UCB). Comparative studies of NK cell differentiation between cultured and freshly isolated HSCs are important in the development of NK cell therapy for cancer. This study aimed to compare the maturation stages of NK cell differentiation between cultured and newly isolated HSC samples using interleukin-2 in the absence of feeder cells. Methods: Differentiation cultures were divided into two groups according to HSC source. Giemsa staining and flow cytometry were performed to determine the maturation stages and the presence of NKp46 receptors, respectively. Results: Giemsa staining revealed that the cultured HSC samples produce a higher number and more mature (stage 5) NK cells than the freshly isolated HSC samples. Flow cytometry showed that the NKp46 mean fluorescence intensity significantly differed between the two samples, and a high level of NKp46 activation receptor was found in the isolated samples on day 35. Conclusions: The cultured HSC samples could produce more mature NK cell populations than the freshly isolated HSCs, which will be beneficial for the therapy applications of NK cells derived from UCB HSCs