Higher number of phospho histone H3 and BrdU-positive mitotic cells were observed in UC-MSC co-cultured cells compared to cells in the OGD group, whereas the reverse effects of HGF and BDNF NAbs on cell mitosis were not significant in histone H3 and BrdU experiments. (UC-MSCs) restore neurological damage through their secretion of paracrine factors. We previously found that UC-MSCs attenuate brain injury by secreting neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and hepatocyte growth factor (HGF). However, how these factors contribute to neuroprotection remains unknown. In this study, we aimed to investigate to what extent UC-MSC-derived HGF and BDNF contribute to neuroprotection using a Transwell co-culture system of neonatal cortical neurons damaged by oxygen-glucose deprivation. The influence of HGF and BDNF were determined by investigating neurons in both the presence and absence of UC-MSCs as DBU these cells consistently secrete both factors and can be blocked by neutralizing antibodies. In the co-culture, UC-MSCs significantly improved neuronal injury, as indicated by an increase in immature neuron number, neurite outgrowth, and cell proliferation. Co-culture of damaged neurons with UC-MSCs also exhibited a reduction in the number of neurons displaying signs of apoptosis/necrosis. The neuroprotective actions of UC-MSCs were partially reverted by neutralizing antibodies. Together, our findings reveal that UC-MSC-secreted HGF and BDNF have neuroprotective effects on damaged neurons. Further studies should address the existence of other potential neurotrophic paracrine factors. in this study. Guo et al. (9) reported the paracrine effects of UC-MSCs on nerve regeneration, observing that UC-MSCs express neurotrophic factors and that UC-MSC-conditioned medium enhances Schwann cell viability TSPAN33 and proliferation via increases in nerve growth factor and BDNF expression. However, since HGF and BDNF are not separately inhibited, there is a possibility that each individual of HGF or BDNF may not contribute to neurotrophic effect. In addition, neuroprotective effect, such as anti-apoptosis/necrosis effect, was not examined. In this study, we DBU focused on whether BDNF and HGF secreted by UC-MSCs exert neuroprotective effect in addition to the neurorestorative effect = 21) were taken from euthanized, pregnant mice in sterile conditions. Fetal brains were removed and cortical tissues were dissected under a microscope. The meninges were then removed and cortical tissues were chopped into small pieces. Cells were dispersed followed by mechanical trituration using Neuron Dissociation Solutions (Wako Pure Chemical Industries, Ltd., Japan) and filtered through a 70 m pore-size cell strainer. Cells were then resuspended in neurobasal medium (GIBCO) supplemented with 2% B27 (Invitrogen) and plated onto Poly-L-Lysine Culture Dishes (BioCoat?, Corning Inc. Japan). Cells were cultured in a humidified incubator at 37C with 5% CO2, and half of the medium was replaced with fresh solution every 3 days. To reduce contamination by glial cells, 10 M cytosine arabinofuranoside (Sigma-Aldrich) was added for 24 h on the 4th day of culture. We cultured cortical neurons for 7days, and OGD procedure was performed. Oxygen-glucose deprived neurons co-cultured with UC-MSCs A model of neonatal cortical neurons injured DBU by OGD was established as previously described (13, 14). For deprivation of glucose, primary cortical neurons were washed twice with phosphate-buffered saline (PBS) and cultured in glucose-free Dulbecco modified eagle medium (GIBCO). Cells were incubated in an anaerobic chamber (95% N2, 5% CO2) (ASTEC Co, Ltd., Japan) at 37C. The OGD condition was maintained for 4 h, after which cells were re-oxygenated in the original medium and placed in a normoxic chamber (37C, 5% CO2). After injury by OGD was completed, co-culture with UC-MSCs was started immediately. Neurons were co-cultured with UC-MSCs according to previously reported methods (10). Briefly, using a 24-well trans-well chamber (Corning, USA) equipped with an 8-m filter membrane, cortical neurons were cultured in the bottom chamber, while UC-MSCs were plated in the upper chamber at 5 104 cells/well overnight for 24 h at 37C with 5% CO2. In experiments aimed to DBU measure HGF DBU and BDNF concentrations in the culture supernatant, MEM without fetal bovine serum was used during co-culture. Multiplex flow cytometric beads assay For HGF and BDNF inhibition, the following neutralizing antibodies (NAbs) were added to UC-MSC culture media in order to deplete HGF and BDNF, as previously reported (12, 15, 16): anti-HGF neutralizing antibody (ab10678, Abcam) and recombinant human TrkB Fc chimera protein (#688-TK; R&D Systems). Briefly, cells were treated with 0.5 g/mL of anti-HGF antibody or 1C2 g/mL recombinant human TrkB Fc chimera protein at plating; media was not changed during the course of the experiment. As a negative.