Figure 7. Blocking PI3K signaling increases motor neuron differentiation in hNSCs grafted into adult rat spinal cords.

(A–B) Representative confocal images of enhanced green fluorescent protein (eGFP)-labeled hNSCs in the dorsal horn of the adult rat spinal cord L4 at 1 month post transplantation. White curve outlines the dorsal horn in (A). (B), higher magnification images of the inset in (A). Grafted hNSCs with FL-priming alone did not co-label with choline acetyltransferase (ChAT, red) or HB9 (blue). (C–D) FL-primed hNSCs, when treated with LY294002, differentiated into Hb9/ChAT immunoreactive motor neurons in the Lamina VII of rat spinal cord. (D), higher magnification images of the inset in (C). (E) FL-primed hNSCs differentiated into ChAT⁺/Hb9⁺ motor neurons in the ventral horn. (F) FL-primed and LY294002-treated hNSCs differentiated into Hb9⁺/ChAT⁺ motor neurons in the ventral horn. (G) Cell counts show a significant increase of the percent of ChAT⁺ neurons from the grafted eGFP⁺ hNSCs treated with LY294002 when compared to the control. Scale bars = 50 µm. C, central canal. Arrow, grafted eGFP⁺ hNSCs become Hb9⁺/ChAT⁺ motor neurons. Arrowhead, endogenous Hb9⁺/ChAT⁺ motor neurons. (H) Proposed model for differential effects of FGF and EGF on motor neuron differentiation from hNSCs. FGF and EGF, via activating their receptor tyrosine kinases (FGFR and EGFR respectively), differentially affect the PI3K/Akt/GSK3β pathway. FGFR-induced PI3K/Akt activation is lower than EGFR, which results in a lower phosphorylation of GSK3β at Ser 9 with increased activity. Higher GSK3β activity leads to an enhanced transcription of HB9, presumably via Ngn2 phosphorylation/activation. PKC-ζ cross-talks with Akt through mutual inhibition, which then modulates GSK3β activity and HB9 expression.