(A) Experimental design. Isolated VM cells were expanded and patterned in vitro prior to transfection to overexpress Wnts. Cell phenotype was examined following in vitro differentiation or transplantation into parkinsonian mice. (B) Morphogens Shh and FGF8 significantly increased the proportion of TH⁺ spheres out of total spheres compared with FGF2 treatment alone. The number of TH⁺ neurons per VMN increased in the presence of morphogens in both passage 1 (C) and passage 2 cultures (G). Wnt overexpression had little effect on TH expression in FGF2-treated VMNs, while Wnt1, and more predominantly Wnt5a, enhanced both the percentage TH⁺ per total spheres and the number of TH neurons per sphere in FGF2/Shh/FGF8 VMNs (C and D). (E) Percent Nurr1⁺ spheres significantly increased compared with FGF2-treated spheres in response to Shh and FGF8 as well as Wnt proteins. Note that Wnt1 increased the percentage of Nurr1⁺ spheres (E) but not TH⁺ spheres per total (B), suggesting lack of specificity of proliferation in all precursor cells, while Wnt5a increased both Nurr and TH/Tuj1/βIII-tubulin, indicating selective increased differentiation of Wnt5a-FGF2/Shh/FGF8–treated VMNs. (F) Photomicrographs of VMNs treated with FGF2 or FGF2/Shh/FGF8 and Wnt1 or Wnt5a. (G) Similar trends in the regulation of TH⁺ cell numbers were noted in passage 2 cultures compared to passage 1; however, the percentage of TH⁺ cells per sphere was reduced with subsequent passaging. (H) Clonal analysis of VMN cells identified multipotent sphere-initiating neural stem cells that gave rise to neurons (Tuj1/βIII-tubulin), astrocytes (GFAP), and oligodendrocytes (O4) after differentiation. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 200 μm (F); 100 μm (H).