Neurotrophic Factor Secretion and Neural Differentiation Potential of Multilineage-differentiating Stress-enduring (Muse) Cells Derived from Mouse Adipose Tissue

Multilineage-differentiating stress-enduring (Muse) cells are endogenous pluripotent stem cells that can be isolated based on stage-specific embryonic antigen-3 (SSEA-3), a pluripotent stem cell-surface marker. However, their capacities for survival, neurotrophic factor secretion, and neuronal and glial differentiation are unclear in rodents. Here we analyzed mouse adipose tissue-derived Muse cells in vitro. We collected mesenchymal stem cells (MSCs) from C57BL/6 J mouse adipose tissue and separated SSEA-3(+), namely Muse cells, and SSEA-3(-), non-Muse cells, to assess self-renewability; pluripotency marker expression (Nanog, Oct3/4, Sox2, and SSEA-3); spontaneous differentiation into endodermal, mesodermal, and ectodermal lineages; and neural differentiation capabilities under cytokine induction. Neurally differentiated Muse and non-Muse cell functions were assessed by calcium imaging. Antioxidant ability was measured to assess survival under oxidative stress. Brain-derived neurotrophic factor (BDNF), vascular endothelial cell growth factor (VEGF), and hepatocyte growth factor (HGF) secretion were analyzed in enzyme-linked immunosorbent assays. SSEA-3(+) Muse cells (6.3 +/- 1.9% of mouse adipose-MSCs), but not non-Muse cells, exhibited self-renewability, spontaneous differentiation into the three germ layers, and differentiation into cells positive for Tuj-1 (27 +/- 0.9%), O4 (17 +/- 3.4%), or GFAP (23 +/- 1.3%) under cytokine induction. Neurally differentiated Muse cells responded to KCl depolarization with greater increases in cytoplasmic Ca2+ levels than non-Muse cells. Cell survival under oxidative stress was significantly higher in Muse cells (50 +/- 2.7%) versus non-Muse cells (22 +/- 2.8%). Muse cells secreted significantly more BDNF, VEGF, and HGF (273 +/- 12, 1479 +/- 7.5, and 6591 +/- 1216 pg/mL, respectively) than non-Muse cells (133 +/- 4.0, 1165 +/- 20, and 2383 +/- 540 pg/mL, respectively). Mouse Muse cells were isolated and characterized for the first time. Muse cells showed greater pluripotency-like characteristics, survival, neurotrophic factor secretion, and neuronal and glial-differentiation capacities than non-Muse cells, indicating that they may have better neural-regeneration potential.