Regenerative activities of signaling-selective 3K3A-APC for human NSCs. (A-C) 3K3A-APC stimulates neuronal proliferation and differentiation from human embryo-derived NSCs in vitro. (A) Stimulation of human NSCs proliferation in culture by 3K3A-APC requires PAR1 and PAR3 but not PAR2. Quantification of proliferation was based on the percentage of Ki-67⁺/nestin⁺ cells in culture after 48 hours in the presence and absence of 3K3A-APC (2 nM) and/or PAR1-, PAR2-, and PAR3-specific cleavage site blocking antibodies. (B-C) Enhancement of neuronal proliferation and differentiation of NSCs by 3K3A-APC requires activation of Akt. Activation of the PI3K/Akt pathway by human recombinant 3K3A-APC (2 nM) in human NSCs requires PAR1 and PAR3 (B) and SphK-1 but not SphK-2 (C). Phosphorylation of Akt at Ser473 (pAkt) and total Akt was determined 3 hours after 3K3A-APC or vehicle treatment by western blot in whole-cell extracts of NSCs transfected with PAR1, PAR3, SphK-1, SphK-2, or control small interfering RNA. Intensity of pAkt signal was determined by scanning densitometry and normalized to total Akt. Data are shown as mean ± SEM, n = 3 independent cultures in triplicate. Statistical significance was determined by 1-way analysis of variance followed by Tukey’s post hoc test. NS, not significant. (For details regarding panels A-C, see Guo et al⁷⁰ ). (D) Schematic overview of APC’s regenerative activities for human NPCs. Activation of the PI3K/Akt pathway in NSCs by 3K3A-APC requires PAR1, PAR3, S1P1, SphK-1, and EPCR. Through integration of signaling linked to multiple downstream effectors, activation of the PI3K/Akt signaling node by 3K3A-APC induces proliferation, migration, survival, and differentiation of NSCs. SphK-1, sphingosine kinase-1; SphK-2, sphingosine kinase-2.