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.
Conditioned medium has now gained increasing interest since the development of secretome-based therapy. Various types of cells have been studied as a source of the secretome. One of them is neural progenitor cells (NPCs). These are cells that capable of differentiating into neurons as well as glial cells. Indeed, the study on NPCs has risen in the last few decades, but the study on the differentiated cells has not clearly described. The most common procedures that widely used to get the conditioned medium is starvation. However, cell starvation may cause environmental stress and become an apoptotic trigger for the cells. In this study, we analyzed the effect of starvation on differentiated cells from E17 rat neural progenitor cells (NPCs) based on cells characteristics and secretome profile. We found that starvation decreased cells viability and affected the heterogeneity of the cell population. Astrocytes survived more under nutrient deprivation conditions, and the progenitor cells showed a higher tendency to differentiate to glial cells than neurons. Duration of starvation also influenced the secretome profile, alterations found in protein types and also their function in the biological process. During 24 hours of starvation, cells secreted proteins that were used to maintain cell growth, stimulate differentiation, and produce energy, but there were also proteins that identified and involved in autophagy activation. After 48 hours of starvation, astrocytes that became the dominant cells secreted proteins that try to keep protecting the remaining neurons.
Mesenchymal stem cells (MSCs) are multipotent cells and can differentiate into neurons and glial cells. In vitro differentiation would be done by the addition of various factors. There remains no comparison for the differentiation of MSCs from rat bone marrow (rBMMSCs) and adipose tissue (rATMSCS) into neurons and glial cells with basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and brain‐derived neurotrophic factor (BDNF). The aims of this study were to investigate the effect of bFGF, EGF, and BDNF supplementation on the differentiation ability of rBMMSCs and rATMSCs into neurons and glial cells. MSCs were cultured with bFGF and EGF for 4 days and then BDNF was added until day 8. Characterization of MSCs before and after induction was carried out by observing the cell morphology and several cell markers. Flowcytometry analysis was performed for MSCs markers (CD90, CD29) and neurons and glial cell markers (A2B5, Beta‐III‐tubulin, PSAN‐CAM); while MAP‐2, a neuron marker, was analyzed by immunocytochemistry. Induction of both types of MSCs showed MAP‐2‐positive cells, decreased MSCs markers, and in rBMMSCs showed increased neuron markers. The number of neuron marker positive cells in rBMMSCS was higher than rATMSCs. This study showed that the addition of bFGF, EGF, and BDNF to the medium induced rBMMSCs into neurons and glial cells, but the conditions were not optimal for rATMSC as judged by the expression of neural markers (A2B5, Beta‐III‐tubulin, PSAN‐CAM, and MAP‐2).
Objective: Rat embryonic fibroblasts (REFs) and rat bone marrow-derived mesenchymal stem cells (rat-BMMSCs) an be used as in vitro models for a variety of studies, including for degenerative diseases such as arterial ischemia, tissue engineering and development of induced pluripotent stem cells (iPSCs). Therefore, the further developments of the use of these two cells of great importance. Methods: The experiments were performed with Wistar rat, those with 15-17 d gestation (aged 32 w) as a REFs source and those aged 12 w as a BMMSCs source. Dulbecco's modified eagle medium (DMEM) was used for both cell cultures but with different media supplements. Proliferation ability was determined for both by calculating population doubling time (PDT). Characterization was performed by differentiation testing into osteocyte, chondrocyte and adipocyte cells by staining with Alizarin Red, Alcian Blue and Oil Red O and by an investigation of specific antigen characteristics using flow cytometry with positive CD90 and CD29 and negative CD34 markers. Results: Morphologically, the REFs and rat-BMMSCs had the same fibroblasts like shape. PDT was higher for the REFs than the BMMSCs (p<0.05), and both could differentiate into osteocytes, chondrocytes and adipocyte. The characteristics of the positive markers (CD29 and CD90) were higher in rat-BMMSCs than in REFs. Conclusion: In this study demonstrated that the explant method for REFs isolation and flushing method for rat-BMMSC isolation are both effective. It also showed that rat-BMMSC grow faster than REFs, and that both cells have the same differentiation ability as rat-BMMSCs but with different specific surface antigen characteristics.
Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into osteocyte, chondrocyte, and adipocyte. MSCs are promising candidates for a regenerative disease therapy because of their ability to selfrenewal and multi-lineage differentiation. MSCs can be isolated from bone marrow (BM), adipose tissues, Wharton jelly, placenta, dermis, and dental pulp. The aim of this study was to compare isolation of bone marrow MSCs from femur and tibia of Swiss Webster mice. It was an in vitro laboratory experimental design. Mice BMMSCs were isolated from mice femur and tibia bone marrow with flushing methods. Mice femur and tibia were immersed in povidone-iodine solution and alcohol 70 % to bones decontaminated and removed the attached fat and connective tissue. Both ends of the bone are cut and flushing with culture medium. The cells suspension incubated in 5 % CO2 incubator. Characterization of mBMMSCs was done by observed cells number. Differentiation potency of MSCs into osteocyte, chondrocyte, and adipocyte were tested using the commercially available kit with staining by alizarin red, alcian blue and oil red O. SPSS with independent t-test. The results showed that cells number of mice BMMSCs after first passage from tibia is higher than femur. Both of mice MSCs from femur and tibia can differentiate to osteocyte, chondrocyte, and adipocyte. mBMMSCs can be isolated from femur and tibia with same ability of differentiation to osteocyte, chondrocyte, and adipocyte.
Background Conditioned medium is the medium obtained from certain cultured cells and contained secretome from the cells. The secretome, which can be in the form of growth factors, cytokines, exosomes, or other proteins secreted by the cells, can induce the differentiation of cells that still have pluripotent or multipotent properties. Objectives This study examined the effects of conditioned medium derived from E17 rat brain cells on cells with pluripotent properties. Methods The conditioned medium used in this study originated from E17 rat brain cells. The CM was used to induce the differentiation of primary colonies of mice blastocysts. Primary colonies were stained with alkaline phosphatase to analyze the pluripotency. The morphological changes in the colonies were examined, and the colonies were stained with GFAP and Neu-N markers on days two and seven after adding the conditioned medium. Results The conditioned medium could differentiate the primary colony, beginning with the formation of embryoid-body-like structure; round GFAP positive cells were identified. Finally, neuron-like cells testing positive for Neu-N were observed on the seventh day after adding the conditioned medium. Conclusions Conditioned medium from different species, in this case, E17 rat brain cells, induced and promoted the differentiation of the primary colony from mice blastocysts into neuron-like cells. The addition of CM mediated neurite growth in the differentiation process.
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