Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice
Clare L. Parish, … , Olle Lindvall, Ernest Arenas
Clare L. Parish, … , Olle Lindvall, Ernest Arenas
Published December 3, 2007
Citation Information: J Clin Invest. 2008;118(1):149-160. https://doi.org/10.1172/JCI32273.
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Research Article

Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice

Abstract

Dopamine (DA) cell replacement therapy in Parkinson disease (PD) can be achieved using human fetal mesencephalic tissue; however, limited tissue availability has hindered further developments. Embryonic stem cells provide a promising alternative, but poor survival and risk of teratoma formation have prevented their clinical application. We present here a method for generating large numbers of DA neurons based on expanding and differentiating ventral midbrain (VM) neural stem cells/progenitors in the presence of key signals necessary for VM DA neuron development. Mouse VM neurospheres (VMNs) expanded with FGF2, differentiated with sonic hedgehog and FGF8, and transfected with Wnt5a (VMN-Wnt5a) generated 10-fold more DA neurons than did conventional FGF2-treated VMNs. VMN-Wnt5a cells exhibited the transcriptional and biochemical profiles and intrinsic electrophysiological properties of midbrain DA cells. Transplantation of these cells into parkinsonian mice resulted in significant cellular and functional recovery. Importantly, no tumors were detected and only a few transplanted grafts contained sporadic nestin-expressing progenitors. Our findings show that Wnt5a improves the differentiation and functional integration of stem cell–derived DA neurons in vivo and define Wnt5a-treated neural stem cells as an efficient and safe source of DA neurons for cell replacement therapy in PD.

Authors

Clare L. Parish, Gonçalo Castelo-Branco, Nina Rawal, Jan Tonnesen, Andreas Toft Sorensen, Carmen Salto, Merab Kokaia, Olle Lindvall, Ernest Arenas

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Figure 1

Expansion and DA differentiation of VMNs.

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Expansion and DA differentiation of VMNs. (A) Experimental design. Isola...
(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).