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. 2021 Mar;10(3):374-384.
doi: 10.1002/sctm.20-0301. Epub 2020 Dec 15.

Brain-derived neurotropic factor mediates neuroprotection of mesenchymal stem cell-derived extracellular vesicles against severe intraventricular hemorrhage in newborn rats

So Yoon Ahn  1   2 , Dong Kyung Sung  2 , Young Eun Kim  2 , Sein Sung  1 , Yun Sil Chang  1   2   3 , Won Soon Park  1   2   3
Affiliations

Brain-derived neurotropic factor mediates neuroprotection of mesenchymal stem cell-derived extracellular vesicles against severe intraventricular hemorrhage in newborn rats

So Yoon Ahn et al. Stem Cells Transl Med. 2021 Mar.

Abstract

Brain-derived neurotropic factor (BDNF), which is secreted by mesenchymal stem cells (MSCs), protects against severe intraventricular hemorrhage (IVH)-induced brain injuries. Although the paracrine protective effects of MSCs are mediated primarily by extracellular vesicles (EVs), the therapeutic efficacy of MSC-derived EVs and the role of the BDNF in the EVs have not been studied. This study aimed to determine whether MSC-derived EVs attenuate severe IVH-induced brain injuries, and if so, whether this protection is mediated by BDNF transfer. We compared the therapeutic efficacy of MSCs, MSC-derived EVs with or without BDNF knockdown, and fibroblast-derived EVs in vitro in rat cortical neuronal cells challenged with thrombin and in vivo in newborn rats by injecting 200 μL of blood at postnatal day (P) 4 and transplanting 1 × 105 MSCs or 20 μg of EVs at P6. The MSCs and MSC-derived EVs, but not the EVs derived from BDNF-knockdown MSCs or fibroblasts, significantly attenuated in vitro thrombin-induced neuronal cell death and in vivo severe IVH-induced brain injuries such as increased neuronal cell death, astrogliosis, and inflammatory responses; reduced myelin basic protein and neurogenesis; led to progression of posthemorrhagic hydrocephalus; and impaired behavioral test performance. Our data indicate that MSC-derived EVs are as effective as parental MSCs in attenuating severe IVH-induced brain injuries, and this neuroprotection is primarily mediated by BDNF transfer via EVs.

Keywords: brain-derived neurotropic factor; extracellular vesicles; intraventricular hemorrhage; mesenchymal stem cells.

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Conflict of interest statement

W.S.P., Y.S.C., D.K.S., and S.Y.A. declared potential conflicts of interest arising from a filed or issued patent titled “Pharmaceutical composition for treating cerebrovascular diseases, containing stem cell‐derived exosome as active ingredient” as coinventors, not as patentees. The other authors declared no potential conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Confirmation of extracellular vesicles (EVs). EVs were isolated from the cell culture media using ultracentrifugation. A, The size and number of EVs as measured using NanoSightNS300 and Nanoparticle Tracking Analysis software. B, Scanning electron micrograph (SEM) of EVs loaded on a polycarbonate membrane. Transmission electron micrograph (TEM) of EVs. EVs on copper grids and stained with uranyl acetate. Representative SEM (left) and TEM (right). C, Representative immunoblot for the organelle marker proteins in mesenchymal stem cell‐derived EVs, CD9, CD63, CD81, cytochrome C for mitochondria, fibrillarin for the nucleus, and GM130 for the Golgi
FIGURE 2
FIGURE 2
Neuroprotective effect of extracellular vesicles on in vitro thrombin‐induced primary cultured rat cortical neurons. A, Representative fluorescence micrographs of the penumbra area with staining for terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) (green, TUNEL‐positive); B, The number of TUNEL‐positive cells captured using fluorescent microscopy; C, Cell viability, expressed as relative proliferation rate (%) to the normal control group; D, Cytotoxicity, expressed as relative lactate dehydrogenase (LDH) release (%) to positive control (100% fully killed cells); E, Malondialdehyde (MDA) level. RatNC, rat neuronal cell normal control; Thr, thrombin treatment; HuMSC, Human MSCs treatment; HuMSC‐EV, extracellular vesicles from naïve human MSCs; HuFibroblast‐EV, extracellular vesicles from human fibroblasts; cont siRNA HuMSC‐EV, extracellular vesicles from human MSCs transfected with scrambled siRNA; BDNF siRNA HuMSC‐EV, extracellular vesicles from human MSCs transfected with BDNF siRNA. Data are presented as mean ± SE of the mean. *P < .05 compared with the rat neuronal cells normal control, #P < .05 compared with the rat neuronal cells treated with thrombin injury control, Φ P < .05 compared with rat neuronal cells treated with thrombin and human MSCs, Ψ P < .05 compared with rat neuronal cells treated with thrombin and human MSCs‐EVs
FIGURE 3
FIGURE 3
BDNF knockdown in the extracellular vesicles abolished the therapeutic effects of MSCs in attenuating ventricular dilatation and its progression after severe intraventricular hemorrhage (IVH). A, Representative serial brain magnetic resonance imaging (MRIs) from each group 1, 7, and 28 days after inducing IVH (at P5, P11, and P32, respectively). B, Ventricle: whole brain volume ratio as measured by MRI. Data are expressed as mean ± SE of the mean. Data are presented as mean ± SE of the mean. *P < .05 compared with the normal control, #P < .05 compared with the IVH injury control, Φ P < .05 compared with IVH + MSCs, Ψ P < .05 compared with IVH + MSCs‐EVs. NC, normal control; IVH, intraventricular hemorrhage control; M, MSCs transplantation; M‐EV, extracellular vesicles from naïve MSCs; Fibro‐EV, extracellular vesicles from fibroblasts; cont siRNA M‐EV, extracellular vesicles from MSCs transfected with scrambled siRNA; BDNF siRNA M‐EV, extracellular vesicles from MSCs transfected with BDNF siRNA
FIGURE 4
FIGURE 4
BDNF knockdown in the extracellular vesicles abolished the therapeutic effects of MSCs in improving brain myelination and in attenuating cell death and reactive gliosis after severe IVH. A, Representative immunofluorescence photomicrographs of the periventricular area with staining for TUNEL (green), glial fibrillary acidic protein (GFAP) (red), ED1 (red), doublecortin (DCX) (red), myelin basic protein (MBP) (green), and 4′,6‐diamidino‐2‐pheylindole (DAPI) (blue). B, The average number of TUNEL and ED1‐positive cells and mean light intensity of GFAP, DCX, and MBP immunofluorescence per high‐power field (HPF) in each group. Data are expressed as mean ± SE of the mean. Data are presented as mean ± SE of the mean. *P < .05 compared with the normal control, #P < .05 compared with the IVH injury control, Φ P < .05 compared with IVH + MSCs, Ψ P < .05 compared with IVH + MSCs‐EVs. NC, normal control; IVH, intraventricular hemorrhage control; M, MSCs transplantation; M‐EV, extracellular vesicles from naïve MSCs; Fibro‐EV, extracellular vesicles from fibroblasts; cont siRNA M‐EV, extracellular vesicles from MSCs transfected with scrambled siRNA; BDNF siRNA M‐EV, extracellular vesicles from MSCs transfected with BDNF siRNA
FIGURE 5
FIGURE 5
BDNF knockdown in the extracellular vesicles abolished the therapeutic effects of MSCs in downregulating brain inflammation after severe IVH. Levels of inflammatory cytokines including interleukin (IL)‐1α, IL‐β, and IL‐6 and tumor necrosis factor (TNF)‐α in brain tissue homogenates, A, and cerebrospinal fluid, B, on P32. Data are expressed as mean ± SE of the mean. Data are presented as mean ± SE of the mean. *P < .05 compared with the normal control, #P < .05 compared with the IVH injury control, Φ P < .05 compared with IVH + MSCs, Ψ P < .05 compared with IVH + MSCs‐EVs. NC, normal control; IVH, intraventricular hemorrhage control; M, MSCs transplantation; M‐EV, extracellular vesicles from naïve MSCs; Fibro‐EV, extracellular vesicles from fibroblasts; cont siRNA M‐EV, extracellular vesicles from MSCs transfected with scrambled siRNA; BDNF siRNA M‐EV, extracellular vesicles from MSCs transfected with BDNF siRNA
FIGURE 6
FIGURE 6
BDNF knockdown in the extracellular vesicles abolished the therapeutic effects of MSCs in improving behavioral function after severe IVH. Sensorimotor functional outcomes on the negative geotaxis test, A, and rotarod test, B, on P32. Data are expressed as mean ± SE of the mean. Data are presented as mean ± SE of the mean. *P < .05 compared with the normal control, #P < .05 compared with the IVH injury control, Φ P < .05 compared with IVH + MSCs, Ψ P < .05 compared with IVH + MSCs‐EVs. NC, normal control; IVH, intraventricular hemorrhage control; M, MSCs transplantation; M‐EV, extracellular vesicles from naïve MSCs; Fibro‐EV, extracellular vesicles from fibroblasts; cont siRNA M‐EV, extracellular vesicles from MSCs transfected with scrambled siRNA; BDNF siRNA M‐EV, extracellular vesicles from MSCs transfected with BDNF siRNA
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