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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.

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.

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).

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