304 - Neuroprotective Effect of Extracellular Vesicles derived from BDNF Overexpressing-TERT Immortalized Mesenchymal Stromal Cells in Neonatal Intraventricular Hemorrhage Model

Publication Number: 4298.304

Young Eun Kim, Samsung medical center, Gangnam-gu, Seoul-t'ukpyolsi, Republic of Korea; Yun Sil Chang, samsung medical center, Gangnam-gu, Seoul-t'ukpyolsi, Republic of Korea; So Yoon Ahn, samsung medical center, gangnamgu, Seoul-t'ukpyolsi, Republic of Korea; So yeon Jung, Samsung medical center, Gangnamgu, Seoul-t'ukpyolsi, Republic of Korea; yuna Bang, Samgsung medical center, Gangnam, Seoul-t'ukpyolsi, Republic of Korea; Misun Yang, Samsung Medical Center, seoul, Seoul-t'ukpyolsi, Republic of Korea; Se In Sung, Samsung Medical Center, seoul, Seoul-t'ukpyolsi, Republic of Korea

Background: We previously demonstrated that brain derived neurotropic factor (BDNF) knockdown in mesenchymal stem cells (MSC)-derived extracellular vesicles (EVs) led to a reduction in their therapeutic efficacy, indicating that BDNF is a key effector molecule responsible for the therapeutic effects of MSC-EVs

Objective: In the present study, we aimed to determine whether overexpression of BDNF enhances the therapeutic efficacy of MSC-derived EVs in vitro and in vivo model of neonatal IVH.

Design/Methods: The exogenous BDNF gene was introduced into TERT-immortalized human Wharton's jelly-derived MSCs via retroviral transduction. EVs were isolated using tangential flow filtration from 24-hour serum-free conditioned media. TERT-immortalized MSCs without BDNF overexpression were used as control MSC. In vitro IVH models were established by exposing primary cultured rat neurons and BV2 microglia to 40 U of thrombin for 24 hours. In vivo severe IVH was induced by infusing 300 µL of maternal rat blood into the lateral ventricles of postnatal day 4 (P4) rat pups. At P6, naïve-EVs or BDNF-EVs (2 × 10⁸ particles in 0.01 mL) were administered intracerebrally. After four weeks of follow-up MRI and behavioral tests, brain tissues and cerebrospinal fluid (CSF) were harvested at P32 for analysis.

Results: In the in vitro model, thrombin treatment increased cytotoxicity-as evidenced by decreased cell viability, elevated LDH release, and higher MDA levels-as well as the expression of inflammatory cytokines (IL-6 and TNF-α) compared with normal controls. However, treatment with BDNF-EVs markedly reversed these changes, exhibiting significantly greater cytoprotective and anti-inflammatory effects than naïve EVs.
In the in vivo IVH model, posthemorrhagic ventricular dilatation confirmed with in-vivo MRI was not atteunauted by naïve EV treatment but was significantly reduced following BDNF-EV. In the periventricular zone, IVH-induced increases in the density of GFAP-positive astrocytes, as well as the numbers of ED-1-positive microglia and TUNEL-positive cells, were significantly attenuated following treatment with BDNF-overexpressing EVs compared with naïve EVs. Consistently, BDNF-EVs reduced IL-1α and IL-6 levels in the CSF, demonstrating a greater anti-inflammatory effect than naïve EVs. In the passive avoidance test, memory performance was significantly improved in the BDNF-EV group compared with the naïve EV group.

Conclusion(s): BDNF-EV exhibited enhanced neuroprotective effects compared to naïve EV, improving neuronal survival and memory function while reducing inflammation and ventriculomegaly in a neonatal IVH model.

Reduced ventricular dilatation, reactive gliosis, activated microglia and cell death induced by severe IVH observed after BDNF overexpressing-EVs
(A) Serial brain magnetic resonance images (MRI) of the three groups and ventricle-to-whole brain volume ratio. (B) Immnunofluorescence micrographs and quantified averages of the ventricular area with GFAP (red; upper panel), ED1 (green; mid panel), and TUNEL (greeen; bottom panel) staining. Data are expressed as means ± SEM.