FCM analysis of ENSc at T0 and T7. (A) As reported by FCM analysis, freshly isolated ENSc (T0) resulted as a heterogeneous population including stem-like, progenitor, glial, and neuronal cells. For each experimental condition, 104 cells were used for acquisition by FACSCanto II and data were expressed as percentage (%) of positive cells ± standard deviation (SD) where each marker was compared to control samples stained only with isotype or secondary control antibody. The positive expression was quantified using the overton subtraction tool of Summit 4.3 software (Beckman Coulter Inc). Under SM and BM culture conditions, the aspecific modulation of glial and neuronal differentiation was detected. (B) Immunophenotypical characterization by FCM of ENSc cultured for 7 days (T7). Data were expressed as percentage (%) of positives (black profile) ± SD for each marker compared to corresponding staining control (gray profile). (C) FCM analysis of PAN neuronal, Frizzled 9, and TLR4 expression. Data were reported as FSC vs fluorescent marker dot plot where R1 (blue colored) and R2 (black colored) subsets were defined. The positive expression of each target marker ± SD was discriminated in gate G1 defined with respect to staining control.