Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model

Abstract

Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ES cells are currently the most promising donor cell source for cell-replacement therapy in PD. We previously described a strong neuralizing activity present on the surface of stromal cells, named stromal cell-derived inducing activity (SDIA). In this study, we generated neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons. We demonstrated that FGF20, preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ES cell-derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms.

Figure 1

Neural progenitors induced from primate ES cells. (A) Time course of neural progenitor marker expression in monkey ES cells cultured on PA6 cells. (B) Detached ES cell colonies formed spheres similar to those of neural progenitor cells. (C–E) Spheres were immunoreactive for NCAM (C, green), Musashi-1 (D, red), and Nestin (E, green). Scale bar: 100 μm.

Figure 2

Expression of differentiated neural and neuronal subtype markers. Differentiated spheres were stained with antibodies against TuJ1 (A and B, green; E–H, blue), GFAP (B, red), Map2ab (C and D, green), GalC (D, red), GABA (E, red), glutamate (Glu; F, green), serotonin (Ser; G, red), and ChAT (H, green). Scale bar: 100 μm. The proportions of cells expressing differentiated neural (I) and neurotransmitter-related (J) markers are expressed as the mean ± SD of 3 independent cultures.

Figure 3

DA neurons differentiated from ES cell–derived neurospheres. (A–D) Differentiated spheres treated with FGF2 and FGF20 were stained with antibodies against TH (red) and TuJ1 (green). Scale bars: 50 μm. (E) The proportion of TH-positive cells to TuJ1-positive cells is expressed as the mean ± SD of 3 to 5 independent cultures. *P < 0.05. (F) RT-PCR for mesencephalic DA neuron markers Pax2, Ptx3, Nurr1, Lmx1b, and TH in cells treated with FGF2 and FGF20 (left) or FGF2 and EGF (right). (G) HPLC measuring concentration of dopamine released by SDIA- and FGF2- and FGF20-treated monkey ES cells in response to high K⁺ depolarizing stimuli (blue line). Dopamine standard, red line.

Figure 4

Function of ES cell–derived neurospheres in MPTP-treated monkeys. Behavioral scores (A) and PET study (B and C) of ES cell–transplanted (n = 6) and sham-operated animals (n = 4). (B) Mean Ki values from entire putamen. (C) Increased ¹⁸F-fluorodopa uptake in the putamen of ES cell–transplanted animals. All values are mean ± SD. *P < 0.05.

Figure 5

Survival of ES cell–derived DA neurons in the striatum. (A–C) Grafted cells (BrdU-labeled, green) survived and differentiated into DA neurons (TH-positive, red) along the needle tract (merged image C). Scale bar: 500 μm. (D–I) Colocalization (arrows in F and I) of BrdU (D, F, G, and I, green) and TH (E and F, red) or DAT (H and I, red) shows that graft-derived cells have dopaminergic character. Scale bar: 50 μm.