F Hes1 [14]. In agreement with this observation, our previous studies using human fetal cortical NPC culture have suggested that TNF-a induced astrogliogenesis and inhibited neurogenesis via the Jak-STAT3 pathway [18]. The Jak-STAT3 signaling is a critical component of the astrogliogenic machinery during brain development [20,25]. Our previous study demonstrated that STAT3 is involved in TNF-ainduced astrogliogenesis and inhibition of neurogenesis [18]. However, it is not likely that TNF-a is a direct upstream effector for STAT3 activation in human NPCs, as TNF-a treatment did not induce immediate phosphorylation of STAT3 (at 30 min). Instead, TNF-a activated STAT3 at delayed time points (6 h and 24 h) (Figure 1). Meanwhile, conditioned medium collected from TNF-a-treated NPCs induced immediate STAT3 activation at 30 min, but not at 6 h and 24 h, suggesting that TNF-a activates STAT3 indirectly through induction of upstream regulators of STAT3. Members of the IL-6 cytokine family such as, LIF, IL-6 and CNTF, are able to activate the Jak-STAT signaling pathway and promote astroglial 4 IBP differentiation [20,21]. We have detected IL-6 family cytokine expression in NPCs upon TNF-a treatment and 
found that TNF-a dramatically increased the mRNA expression of IL-6 and LIF but not CNTF (Figure 2). Using neutralizing antibodies, LIF was identified as the molecule responsible for the preferential differentiation of NPCs toward astrocytic lineage (Figure 4,5,6).LIF signals through the heterodimeric complex of common glycoprotein 130 (gp130) and LIF receptor (LIFR) subunits. A number of studies have shown that gp130/LIFR-mediated signaling has pleiotropic action on different cell types. LIF is well known for promoting mouse embryonic stem (ES) cell self-renewal [26] and the maintenance/self-renewal of cultured mouse and human embryonic NSCs [27?1]. In addition, LIF-induced JakSTAT signaling is critical for promoting astrocytic differentiation [20,32,33]. LIF is abundantly secreted from rat cortical neural precursor cells and serves as an autocrine/paracrine factor for the survival and astrocytic differentiation of embryonic cortical precursor cells 23727046 [33]. In our study, we further demonstrated that TNF-a increased LIF production in human NPCs and LIF is responsible for TNF-a-induced STAT3 activation and the sequential astrogliogenesis. In this study, we also observed the up-regulated expression of IL-6 in NPC. However, neutralizing antibody for IL-6 failed to block TNF-a-induced activation of STAT3, suggesting IL-6 may not be the main contributor for TNF-a-induced STAT3 activation and astrogliogenesis. This may be due to the lower expression of IL-6 receptor on NPCs (less than 1 of LIF receptor mRNA values, data not shown). In conclusion, we have demonstrated that TNF-a activates STAT3 and promotes astrogliogenesis through the autocrine secretion of LIF. This study provides a novel mechanism by which pro-inflammatory cytokines affect neurogenesis and regulate the fate of NPCs. This information is get 1948-33-0 important for developing transplantation therapies or promoting activation of endogenous NPCs for repair in neurodegenerative diseases with brain inflammation.AcknowledgmentsWe kindly acknowledge Dr. Tsuneya Ikezu and Ms. Li Wu who provided technical support for this work. Dr. Yunlong Huang, Mrs. Kristin Leland Wavrin and Ms. Danielle F. Tate provided valuable comments and suggestions about the manuscript.Author ContributionsConceived and designed the experimen.F Hes1 [14]. In agreement with this observation, our previous studies using human fetal cortical NPC culture have suggested that TNF-a induced astrogliogenesis and inhibited neurogenesis via the Jak-STAT3 pathway [18]. The Jak-STAT3 signaling is a critical component of the astrogliogenic machinery during brain development [20,25]. Our previous study demonstrated that STAT3 is involved in TNF-ainduced astrogliogenesis and inhibition of neurogenesis [18]. However, it is not likely that TNF-a is a direct upstream effector for STAT3 activation in human NPCs, as TNF-a treatment did not induce immediate phosphorylation of STAT3 (at 30 min). Instead, TNF-a activated STAT3 at delayed time points (6 h and 24 h) (Figure 1). Meanwhile, conditioned medium collected from TNF-a-treated NPCs induced immediate STAT3 activation at 30 min, but not at 6 h and 24 h, suggesting that TNF-a activates STAT3 indirectly through induction of upstream regulators of STAT3. Members of the IL-6 cytokine family such as, LIF, IL-6 and CNTF, are able to activate the Jak-STAT signaling pathway and promote astroglial differentiation [20,21]. We have detected IL-6 family cytokine expression in NPCs upon TNF-a treatment and found that TNF-a dramatically increased the mRNA expression of IL-6 and LIF but not CNTF (Figure 2). Using neutralizing antibodies, LIF was identified as the molecule responsible for the preferential differentiation of NPCs toward astrocytic lineage (Figure 4,5,6).LIF signals through the heterodimeric complex of common glycoprotein 130 (gp130) and LIF receptor (LIFR) subunits. A number of studies have shown that gp130/LIFR-mediated signaling 
has pleiotropic action on different cell types. LIF is well known for promoting mouse embryonic stem (ES) cell self-renewal [26] and the maintenance/self-renewal of cultured mouse and human embryonic NSCs [27?1]. In addition, LIF-induced JakSTAT signaling is critical for promoting astrocytic differentiation [20,32,33]. LIF is abundantly secreted from rat cortical neural precursor cells and serves as an autocrine/paracrine factor for the survival and astrocytic differentiation of embryonic cortical precursor cells 23727046 [33]. In our study, we further demonstrated that TNF-a increased LIF production in human NPCs and LIF is responsible for TNF-a-induced STAT3 activation and the sequential astrogliogenesis. In this study, we also observed the up-regulated expression of IL-6 in NPC. However, neutralizing antibody for IL-6 failed to block TNF-a-induced activation of STAT3, suggesting IL-6 may not be the main contributor for TNF-a-induced STAT3 activation and astrogliogenesis. This may be due to the lower expression of IL-6 receptor on NPCs (less than 1 of LIF receptor mRNA values, data not shown). In conclusion, we have demonstrated that TNF-a activates STAT3 and promotes astrogliogenesis through the autocrine secretion of LIF. This study provides a novel mechanism by which pro-inflammatory cytokines affect neurogenesis and regulate the fate of NPCs. This information is important for developing transplantation therapies or promoting activation of endogenous NPCs for repair in neurodegenerative diseases with brain inflammation.AcknowledgmentsWe kindly acknowledge Dr. Tsuneya Ikezu and Ms. Li Wu who provided technical support for this work. Dr. Yunlong Huang, Mrs. Kristin Leland Wavrin and Ms. Danielle F. Tate provided valuable comments and suggestions about the manuscript.Author ContributionsConceived and designed the experimen.
