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Effective dopamine (DA) neuron differentiation from neural precursor cells (NPCs) is prerequisite for precursor/ stem cell-based therapy of Parkinson's disease (PD). Nurr1, an orphan nuclear receptor, has been reported as a transcription factor that can drive DA neuron differentiation from non-dopaminergic NPCs in vitro. However, Nurr1 alone neither induces full neuronal maturation nor expression of proteins found specifically in midbrain DA neurons. In addition, Nurr1 expression is inefficient in inducing DA phenotype expression in NPCs derived from certain species such as mouse and human. We show here that Foxa2, a forkhead transcription factor whose role in midbrain DA neuron development was recently revealed, synergistically cooperates with Nurr1 to induce DA phenotype acquisition, midbrain-specific gene expression, and neuronal maturation. Thus, the combinatorial expression of Nurr1 and Foxa2 in NPCs efficiently yielded fully differentiated nigral (A9)-type midbrain neurons with clearly detectable DA neuronal activities. The effects of Foxa2 in DA neuron generation were observed regardless of the brain regions or species from which NPCs were derived. Furthermore, DA neurons generated by ectopic Foxa2 expression were more resistant to toxins. Importantly, Foxa2 expression resulted in a rapid cell cycle exit and reduced cell proliferation. Consistently, transplantation of NPCs transduced with Nurr1 and Foxa2 generated grafts enriched with midbrain-type DA neurons but reduced number of proliferating cells, and significantly reversed motor deficits in a rat PD model. Our findings can be applied to ongoing attempts to develop an efficient and safe precursor/stem cell-based therapy for PD. STEM CELLS 2010;28:501-512

In this study, we have attempted to exploit the neurotrophic actions of astrocytes and Nurr1+Foxa2 functions in this cell type to improve the therapeutic outcomes of NPC transplantation using an animal model of PD. Our in vitro assays using a coculture system and conditioned media treatment revealed that astrocytes, especially those cultured from the ventral midbrain (VM), where HNF3β) is a potent cofactor that synergizes the Nurr1-mediated antiinflammatory roles in glia (16). Furthermore, forced coexpression of Nurr1 and Foxa2 (Nurr1+Foxa2) in glia promotes the synthesis and release of various neurotrophic factors, indicating that engineering of Nurr1 and Foxa2 in glia could become a powerful strategy for treating CNS disorders. Representative images of TH + DA neurons differentiated from VM-NPCs in the presence of Ctx-NPCs (control), Ctx-Ast, or VM-Ast. Scale bar: 100 μm. Insets, enlarged images of the boxed areas (original magnification, ×400). (C) DA neuronal yields at differentiation day 6 (D6). (D-H) Morphometric measurement of DA neuron maturity assessed by neurite outgrowth lengths (D), soma size (E), and by the Sholl test (F and G). In the Sholl analysis, the total number of neurite crossings was counted in each circle with the radius increasing in steps of 15 μm (F). The critical value (G) is the radius at which there was a maximum number of neurite crossings. *P < 0.05, significantly different from the control; # P < 0.05, significantly different from Ctx-Ast, 1-way ANOVA. n = 3-5 wells (C) and 100 (D and E) and 25-30 (F and G) TH + cells in each group.(H) Representative TH + neuronal morphologies reconstructed in 3D by Neurolucida. (I-L) Synaptic densities on TH + fibers. (I) Representative confocal images of synapsin + TH + fibers. Scale bar: 25 μm. Puncta positive for the synaptic vesicle-specific markers SV2 (J), synapsin (K), and bassoon (L) on TH + fibers were counted. *P < 0.05, significantly different from the control; # P < 0.05, significantly different from Ctx-Ast. n = 20 TH + fibers for each group. (M) Presynaptic DA release. n = 3 independent cultures. DA levels were measured in the media conditioned in the differentiated cultures for 24 hours (D13-D15) and in cultures evoked by KCl-mediated depolarization for 30 minutes. *P < 0.05; # P < 0.05, 1-way ANOVA. The Journal of Clinical Investigation R E S E A R C H A R T I C L E4 6 5 jci.org Volume 128 Number 1 January 2018 (Supplemental Figure 1B; supplemental material available online with this article; https://doi.org/10.1172/JCI93924DS1), immunocytochemical analyses revealed that major cell populations in the Ctx-and VM-astroglial cultures at day in vitro (DIV) 14 were positive for GFAP (85% and 82%), CD44 (79% and 76 %), GLT-1 (58% and 71 %), and GLAST protein (71% and 77%), respectively (Supplemental Figure 1A), except S100β (20% and 32%), a soluble protein associated with harmful reactivation of astrocytes (22). A few (0.24% ± 0.35% in Ctx-Ast and 0.43% ± 0.39% in VM-Ast, where Ast indicates astrocytes) and none of the cells in these c...

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