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.

Figure 1. Expansion and DA differentiation of VMNs.

(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).

Figure 2. Wnt5a mediates effects on DA differentiation via noncanonical signaling across rodent species.

(A and B) Effects of Wnt5a overexpression were abolished by using a Wnt5a-blocking antibody (W5-ab) or the D4476 CKI inhibitor (CKI), but not the canonical Wnt blocker dickkopf-1 (DKF1). Mouse recombinant Wnt5a protein (r.m.W5) induced effects comparable to those of Wnt5a overexpression in control-transfected spheres. (C and D) Comparable trends for number of TH⁺ cells per sphere (C) and percentage of TH⁺ spheres per Tuj1/βIII-tubulin⁺ spheres (D) were observed in E12.5 rat compared with E10.5 mouse cultures. However, significantly more TH⁺ cells were present in rat cultures than in mouse cultures. Data are mean ± SD (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Tukey post-hoc test.

Figure 3. VMN-Wnt5a grafts show no signs of tumor formation, proliferation, or cell expansion.

Animals grafted with predifferentiated mouse ES cells (mESC) showed immunoreactivity for various markers of proliferating cells types including Oct3/4 (A), PCNA (B), and phospho–histone-3 (C), which were not seen in VMN and VMN-Wnt5a grafts (E–G and I–K, respectively). (D) Additionally, mouse ES cell grafts showed dense regions of nestin⁺ cells. Extremely few nestin⁺ cells were seen in very small clusters in select VMN grafts (H), and isolated nestin⁺ cells were observed in fewer VMN-Wnt5a grafts (L). Scale bar: 100 μm.

Figure 4. Transplantation of Wnt5a-overexpressing VMNs induces behavioral and cellular recovery in parkinsonian mice.

(A) Time course of amphetamine-induced rotational behavior in sham-, VMN-, and VMN-Wnt5a–grafted animals. VMN transplants resulted in significant behavioral improvement at 8 weeks, while VMN-Wnt5a transplants resulted in full restoration of behavior. (B) Confirmation of behavioral improvements following apomorphine-induced rotational testing at 8 weeks. (C) Number of TH⁺ cells within the striatum of grafted animals. (D–F) Photomicrographs in the striatum of grafts from sham-operated (D), VMN-grafted (E), and VMN-Wnt5a–grafted (F) mice. (G) High-power image of DA neurons residing within a VMN-Wnt5a graft showing classical bipolar morphology (arrow). (H) High-power image of a hypertrophied TH⁺ fiber located outside the graft site (arrowhead in F). (I) Area of TH-immunoreactive fibers within the striatum following grafting. (J) Area of fibers per TH cell. Note that regardless of the increase in cell numbers, the density of fibers per cell in VMN-Wnt5a grafts was significantly greater, suggesting neuritogenesis and greater integration into the host tissue. Data are mean ± SD (n = 6 per group). *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA with Tukey post-hoc test. Scale bars: 200 μm (D–F); 50 μm (G); 100 μm (H).

Figure 5. Wnt5a reduces the number of cells required for functional engraftment in PD mice.

Transplantation of twice the number of VMN cells was required to induce similar behavioral and histological recovery as obtained with VMN-Wnt5a grafts (800,000 and 400,000 cells, respectively). Effects are expressed as changes in rotational behavior (A) and TH⁺ cell counts (B). Data are mean ± SD (n = 3–4 per group). *P < 0.05; **P < 0.01, ANOVA with Tukey post-hoc test.

Figure 6. Improved differentiation of Nurr1⁺ DA precursors and expression of midbrain-specific markers by VMN-Wnt5a cells in vivo.

(A) Number of TH⁺ and Nurr1⁺ cells within the grafts and (B) ratio of TH⁺ to Nurr1⁺ cells. Note there is no significant difference between TH⁺ and Nurr1⁺ cell number in VMN-Wnt5a grafts, suggesting that almost all precursor cells (i.e., 88%) adopted a DA fate (see D), unlike in VMN grafts alone, where numerous Nurr1⁺TH^– cells were observed (C, arrows). Data are mean ± SD (n = 6 per group). *P < 0.05; **P < 0.01, Student’s t test. (E) Pitx3/TH and (F) GIRK2/TH staining within the grafts confirmed midbrain phenotype. (G–K) Expression profile of midbrain DA neuron genes involved in development and function, as assessed by quantitative real-time RT-PCR. Both early and late genes in DA development were upregulated in VMN-Wnt5a grafts. Data are mean ± SD (n = 3–4 per group). *P < 0.05; ***P < 0.001, ANOVA with Tukey post-hoc test. Scale bar: 50 μm.

Figure 7. VMN-Wnt5a cells show the electrophysiological properties of midbrain DA neurons.

(A) Example of triple labeling for biocytin, GFP, and TH immunoreactivity of recorded DA cell, as revealed by confocal microscopy. (B–F) No differences between control-transfected cells and cells transfected with Wnt5 were detected with respect to resting membrane potential and spontaneous firing rate (B); action potential threshold, amplitude, and duration (C); and inward rectification on hyperpolarization steps as assessed by current-voltage curves (E; n = 8 cells/group; mean ± SEM). Spontaneous action potential frequencies varied within both groups, ranging from 0.1 to 10 Hz. Arrowhead in C indicates the onset/threshold of the action potential. (D) In those cells that did not discharge spontaneously, action potentials were elicited by depolarizing current injections via patch pipette. (F) Some TH-GFP⁺ cells in both groups received functional excitatory synaptic inputs, as illustrated by sEPSCs recorded at resting membrane potential in voltage-clamp mode. Insets show enlarged sEPSCs corresponding to the boxes on the upper traces.

Figure 8. VMN-Wnt5a cells show midbrain dopaminergic biochemical properties in vitro and in vivo.

(A) In vitro HPLC revealed a significant increase in DA turnover (ratio of DA to HVA) in Wnt5a-transfected cultures. This differentiation was selective for DA neurons, as it had no effect on release or turnover of other neurotransmitters, such as serotonin (not shown). Data are mean ± SD (n = 4 per group). *P < 0.05, 1-way ANOVA with Tukey post-hoc test. (B and C) HPLC revealed significantly increased DA (B) and DOPAC (C) concentration in VMN and VMN-Wnt5a striatal grafts compared with the lesion animals. VMN-Wnt5a grafts showed no significant difference in DA or DOPAC concentration compared with animals with an intact striatum. (D) Animals receiving VMN grafts showed a significant increase in DA turnover (ratio of DOPAC to DA) as a compensatory mechanism for the reduced DA levels, while DA turnover remained unaltered in VMN-Wnt5 grafts. Data are mean ± SEM (n = 6 per group). *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA on Ranks with Dunn’s post-hoc test.