Wan Ahmad Jailani, Wan Nur Tihani (2022) Characterization Of Amniotic Fluid Stem Cells (Afscs)-Derived Neural Stem Cells Exosomes Treated With Ethanolic Extract Of Centella Asiatica. [Project Paper] (Submitted)
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Abstract
The potential of exosomes as acellular therapy for treating neurodegenerative diseases is emerging. Centella asiatica (CA) has been shown to positively mediate production of neurotrophic factors (NF) from stem cells. Previously, we have observed that ethanolic extract of CA (EECA) promoted higher production of NF in the conditioned medium (CM, secretomes) of rat full-term amniotic fluid stem cell line (R3)-derived neural stem cells (NSCs). Therefore, there is a prospect of EECA in enhancing the quality of NSCs-derived exosomes. Objective: We aimed to evaluate and characterize the exosomes isolated from the CM of R3-derived NSCs treated with EECA. Hypothesis: Treatment of EECA promotes transdifferentiation of R3 into NSCs by generating a higher number and good quality neurospheres, and a higher nestin expression. Next, exosomes with appropriate structures can be isolated from the CM of R3-derived NSCs treated with EECA. Methodology: R3 was transdifferentiated into NSCs through a monolayer differentiation assay by culturing 2.6×104 R3 cells/cm2 in Embryoid Body Medium (EBM) with and without treatment for 2 days. This study included four groups: R3 alone (negative control), R3 treated with 1 ug/mL of EECA, 10 ug/mL of EECA, and 50 uM of dBcAMP (positive control). After 2 days, the EBM in all groups was replaced with DMEM/F12 (a resting medium) for 2 days before collecting the CM. On the other hand, the attached NSCs were trypsinized and subjected to form neurospheres confirming the ability of R3 to transdifferentiate into NSCs followed by assessing the quality of neurospheres (size and expression of nestin by immunocytochemistry, ICC). The exosomes were isolated by sequential ultracentrifugation (Beckman Coulter). Later, the structures of the exosomes produced by R3-derived NSCs were observed under JEOL JEM-2100F transmission electron microscopy (TEM) operated at 200 kV, and images were obtained using Gatan Orius software. Results: R3-derived NSCs treated with EECA formed a higher number and good quality neurospheres with diameters of 50-100 μm. These neurospheres exhibited a higher expression of nestin. TEM revealed that exosomes of 50–150 nm were successfully isolated from R3-derived NSCs treated with EECA. Discussion: The formation of neurospheres and the expression of NSC marker, nestin, confirm the successful transdifferentiation of R3 into NSCs. The treatment of EECA positively influences the quality and quantity of R3-NSCs-derived neurospheres. Based on TEM, EECA-enriched exosomal structures were not compromised upon the treatment with EECA, hence further in-depth investigation is worth pursuing. Conclusion: These findings suggest the effect of EECA as the prospective neuroenhancement adjunct in generating quality exosomes essential for acellular therapy against neurodegenerative diseases.
| Item Type: | Project Paper |
|---|---|
| Depositing User: | Ms. Nor Safa'aton Saidin |
| Date Deposited: | 22 Aug 2023 07:04 |
| Last Modified: | 22 Aug 2023 07:04 |
| URI: | http://psaspb.upm.edu.my/id/eprint/1435 |
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