Neuroprotective effect of placenta‐derived mesenchymal stromal cells: role of exosomes

Abstract

We have established early‐gestation chorionic villus–derived placenta mesenchymal stromal cells (PMSCs) as a potential treatment for spina bifida (SB), a neural tube defect. Our preclinical studies demonstrated that PMSCs have the potential to cure hind limb paralysis in the fetal lamb model of SB via a paracrine mechanism. PMSCs exhibit neuroprotective function by increasing cell number and neurites, as shown by indirect coculture and direct addition of PMSC‐conditioned medium to the staurosporine‐induced apoptotic human neuroblastoma cell line, SH‐SY5Y. PMSC‐conditioned medium suppressed caspase activity in apoptotic SH‐SY5Y cells, suggesting that PMSC secretome contributes to neuronal survival after injury. As a part of PMSC secretome, PMSC exosomes were isolated and extensively characterized; their addition to apoptotic SH‐SY5Y cells mediated an increase in neurites, suggesting that they exhibit neuroprotective function. Proteomic and RNA sequencing analysis revealed that PMSC exosomes contain several proteins and RNAs involved in neuronal survival and development. Galectin 1 was highly expressed on the surface of PMSCs and PMSC exosomes. Preincubation of exosomes with anti‐galectin 1 antibody decreased their neuroprotective effect, suggesting that PMSC exosomes likely impart their effect via binding of galectin 1 to cells. Future studies will include in‐depth analyses of the role of PMSC exosomes on neuroprotection and their clinical applications.—Kumar, P., Becker, J. C., Gao, K., Carney, R. P., Lankford, L., Keller, B. A., Herout, K., Lam, K. S., Farmer, D. L., Wang, A. Neuroprotective effect of placenta‐derived mesenchymal stromal cells: role of exosomes. FASEB J. 33, 5836–5849 (2019). http://www.fasebj.org

Placental Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Myelin Regeneration in an Animal Model of Multiple Sclerosis

“…Additionally, PMSCs secrete high levels of BDNF, HGF and VEGF as measured by ELISA ( Figure 2C). Neuroprotection assays were also performed with PMSCs and it was shown that these cells increase total branching points (fold change 2.01), circulatory length (fold change 1.52) and total segment counts (fold change 1.56) in SH-SY5Y apoptotic cells, which aligns with previous study findings [22]. EVs were then isolated from PMSCs that were shown to have both immunomodulatory and neuroprotective properties.…”

“…Neuroprotection assays were performed exactly as previously described [22]. In brief, the SH-SY5Y neuroblastoma cell line was cultured for 24 h. PMSCs were indirectly cultured in hanging well inserts for 24 h. To assess the neuroprotective properties of PMSCs, SH-SY5Y cells were treated with 1 µM staurosporine to induce apoptosis.…”

“…PMSC-derived EVs were isolated as previously described [22]. In brief, EVs were first depleted from FBS by spinning FBS samples at 112,700 G using the L7 Ultracentrifuge (Beckman Coulter, Brea, CA, USA) and a SW28 rotor for 16 h at 4 • C. Supernatants were collected, aliquoted and stored at −20 • C. PMSCs at passage 4 were seeded at 20,000 cells/cm 2 in T175 flasks (Corning Inc.) in 20 mL of medium containing 5% EV-depleted FBS, 20 ng/mL FGF (Advent Bio), 20 ng/mL EGF (Advent Bio), and 1% P/S (Thermo Fischer Scientific) for 48 h at 37 • C, under 5% CO 2 .…”

“…In brief, EVs were first depleted from FBS by spinning FBS samples at 112,700 G using the L7 Ultracentrifuge (Beckman Coulter, Brea, CA, USA) and a SW28 rotor for 16 h at 4 • C. Supernatants were collected, aliquoted and stored at −20 • C. PMSCs at passage 4 were seeded at 20,000 cells/cm 2 in T175 flasks (Corning Inc.) in 20 mL of medium containing 5% EV-depleted FBS, 20 ng/mL FGF (Advent Bio), 20 ng/mL EGF (Advent Bio), and 1% P/S (Thermo Fischer Scientific) for 48 h at 37 • C, under 5% CO 2 . Conditioned medium was collected and EVs were isolated by differential centrifugation exactly as previously described [22]. After the final centrifugation step, EV pellets were resuspended in 10 µL of triple-filtered PBS (GE, Life Sciences) per T175 flask used for the generation of the conditioned medium.…”

“…To characterize EVs, a Western blotting analysis was performed exactly as previously described [22]. In brief, EVs were treated with NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) and heated to 90 • C. The samples were run, transferred, probed with 1:500 dilution of primary antibodies against Alg-2 interacting protein X (ALIX; rabbit polyclonal, Millipore-Sigma), tumor susceptibility gene (TSG101; clone T5701; Millipore-Sigma), CD9 (clone MM2-57; Millipore-Sigma), calnexin (Clone C5C9, Cell Signaling Technology, Danvers, MA, USA), and CD63 (clone TS63; Thermo Fisher Scientific) in 5% nonfat dry milk in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.5% Tween 20 (Millipore-Sigma).…”