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. 2007 Apr;25(4):918-28.
doi: 10.1634/stemcells.2006-0386. Epub 2006 Dec 14.

Persistent dopamine functions of neurons derived from embryonic stem cells in a rodent model of Parkinson disease

Jose A Rodríguez-Gómez  1 , Jian-Qiang Lu, Iván Velasco, Seth Rivera, Sami S Zoghbi, Jeih-San Liow, John L Musachio, Frederick T Chin, Hiroshi Toyama, Jurgen Seidel, Michael V Green, Panayotis K Thanos, Masanori Ichise, Victor W Pike, Robert B Innis, Ron D G McKay
Affiliations

Persistent dopamine functions of neurons derived from embryonic stem cells in a rodent model of Parkinson disease

Jose A Rodríguez-Gómez et al. Stem Cells. 2007 Apr.

Abstract

The derivation of dopamine neurons is one of the best examples of the clinical potential of embryonic stem (ES) cells, but the long-term function of the grafted neurons has not been established. Here, we show that, after transplantation into an animal model, neurons derived from mouse ES cells survived for over 32 weeks, maintained midbrain markers, and had sustained behavioral effects. Microdialysis in grafted animals showed that dopamine (DA) release was induced by depolarization and pharmacological stimulants. Positron emission tomography measured the expression of presynaptic dopamine transporters in the graft and also showed that the number of postsynaptic DA D(2) receptors was normalized in the host striatum. These data suggest that ES cell-derived neurons show DA release and reuptake and stimulate appropriate postsynaptic responses for long periods after implantation. This work supports continued interest in ES cells as a source of functional DA neurons.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

Figures

Figure 1
Figure 1
Expression pattern of midbrain-related markers by progenitors and differentiated neurons generated from embryonic stem cells. (A): Triple immunocytochemical labeling for En1, Lmx1b, and Foxa2 in progenitor cells (day 4, stage IV). (B): Different cell populations expressing different combinations of genes were observed at this stage. The results are shown as mean ± SEM. (C): Triple immunocytochemical labeling in differentiated dopamine neurons (day 10, stage V). TH+ cells express neuronal marker Hu. Foxa2 expression is maintained in differentiated TH+ neurons. En1 is expressed in TH+ neurons, and Ptx3 expression emerges in most of them. Few TH+ neurons expressed calbindin, which did not colocalized with the Ptx3 gene. A fraction of TH+ neurons (42% ± 1%) expressed RALDH1 protein. Some TH± also coexpressed DAT. Scale = 20 μm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; DAT, dopamine transporter; TH, tyrosine hydroxylase.
Figure 2
Figure 2
Behavioral recovery after embryonic stem (ES)-cell-derived dopamine (DA) neuron grafting, phenotype of grafted DA neurons, and restoration of DA terminal field in 6-hydroxy-dopamine lesioned striatum by grafted cells. (A): Grafting of ES cells caused significant recovery of amphetamine-induced rotational behavior. The results are shown as mean ± SEM (n = 5; *, p < .01; #, p < .05 by a two-tailed Student’s t test compared with rats receiving sham treatment). (B): Double immunocytochemical staining of grafted DA neurons performed at the endpoint of the experiment (i.e., 32 weeks after transplantation). Donor origin of grafted DA neurons was confirmed by colabeling with the mouse-specific antigen M2. Some TH+ neurons coexpressed RALDH1, and we also found TH+/calbindin+ neurons. Double-positive neurons for TH and calretinin were present, as well as TH−/calretinin+ cells. Scale = 20 μm. (C): ES cell-derived DA neurons partially restored TH and RALDH1 immunoreactivity in the lesioned side. TH (green) immunostaining is shown for the grafted (left, C1) and nonlesioned (right, C2) sides, and RALDH1 (red) is shown for the grafted (C3) and nonlesioned (C4) sides. DAPI staining is shown in blue. Scale = 1 mm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; [18F]FECNT, 2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane; TH, tyrosine hydroxylase.
Figure 3
Figure 3
Effect of embryonic stem cell-derived dopamine (DA) neuron graft on monoamine levels studied in vivo by microdialysis performed 12 weeks after grafting. (A): Time-course on DA concentration level after 100 mM K+ isosmotic medium, 50 μM nomifensine, and 30 μM amphetamine in the lesioned and nonlesioned sides of sham and grafted animals. (B): Time-course on DA level shown in (A) for lesioned sides in grafted and sham groups is shown at bigger scale. (C): Time-course of DOPAC and HVA concentrations for nonlesioned sides of both sham and grafted animals and for the lesioned side that received DA neurons. The results are shown as mean ± SEM (n = 5; *, p < .01; #, p < .05 by a two-tailed Student’s t test compared with rats receiving the sham treatment). Abbreviations: DOPAC, 3,4-dihydroxyphenylacetic acid; HK, high potassium; HVA, homovanillic acid.
Figure 4
Figure 4
Dynamic positron emission tomography imaging in naïve rats with [18F]FECNT. (A): Approximately 30 minutes after bolus injection of [18F]FECNT (n = 5), activity in cerebellum was stable despite declining concentrations in striatum. (B): After approximately 150 minutes of constant infusion of [18F]FECNT (n = 5), activities in striatum and cerebellum increased in a linear and parallel manner. Specific binding was operationally defined as the difference between striatum and cerebellum and, therefore, became stable after approximately 150 minutes. The plasma concentration of [18F]FECNT separated from radiometabolites (upper curve) after ~170 minutes of infusion was also obtained. The y-axis on left is for brain measurements and is expressed as percentage of the activity infused per hour. The y-axis on right is the plasma measurements of the concentration of [18F]FECNT and is expressed as a percentage of the constant infusion: ([plasma [18F]FECNT dpm/ml]/activity [mCi] infused per hour) × 100. (C): The difference in time activity curves between striatum and cerebellum was confirmed to be specific binding, since methylphenidate displaced [18F]FECNT in striatum to background levels in cerebellum (n = 4). Abbreviations: CBL, cerebellum; [18F]FECNT, 2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane; h, hour; min, minutes; SPEC, specific binding; STR, striatum.
Figure 5
Figure 5
Recovery on dopamine transporter binding after grafting of 6-hydroxy-dopamine lesioned animals performed 24–28 weeks after transplantation. (A): A naïve rat showed bilaterally symmetrical uptake of 2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane ([18F]FECNT) in striata following constant infusion of the radiotracer. (B): A hemiparkinsonian rat receiving sham treatment displayed negligible uptake in the lesioned striatum. (C): Embryonic stem (ES)-cell transplantation partially restored uptake of [18F]FECNT in the lesioned striatum of a hemiparkinsonian rat. (D): Positron emission tomography (PET) and magnetic resonance imaging images of hemiparkinsonian rat with ES-cell transplantation into the left striatum were coregistered to define anatomic structures. (E): ES-cell transplantation increased the ratio of activity in lesioned/nonlesioned striatum determined by PET imaging with [18F]FECNT. The ratio was greatly reduced in hemiparkinsonian rats that had either no intervention (n = 4) or sham treatment (n = 5). This reduced ratio significantly recovered after transplanting ES-cells into the lesioned striatum of hemiparkinsonian rats (n = 5; *, p < .01 by a two-tailed Student’s t test compared with rats receiving the sham treatment). The horizontal line on each group shows the mean value.
Figure 6
Figure 6
Recovery on 2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane ([18F]FECNT)-specific binding after grafting of 6-hydroxy-dopamine lesioned animals. Time activity curves following constant infusion of [18F]FECNT in rats with (A) sham treatment and (B) embryonic stem (ES) cell transplantation. After 120–150 minutes of constant infusion, uptake in both striatum and cerebellum ascended linearly, and specific binding (SPEC = STR – CBL) became stable. Specific binding in the lesioned striatum displayed a minimal level in rats receiving sham treatment ([A]; n = 5) but increased to approximately one-third the level of nonlesioned striatum after ES cell transplantation ([B]; n = 5). The y-axis is expressed as percentage of the activity infused per hour. Results are shown as mean ± SEM. (C): Distribution volumes of [18F]FECNT-specific binding in striata of hemiparkinsonian rats. The distribution volumes of the nonlesioned striata were similar in sham (n = 5) and ES cell transplanted (n = 5) animals. In contrast, the distribution volumes of the lesioned striata were significantly higher in transplanted than in sham animals (6.71 ± 1.7 vs. 1.15 ± 0.23; *, p < .01 by a two-tailed Student’s t test compared with rats receiving the sham treatment). The horizontal line on each group shows the mean value. Abbreviations: CBL, cerebellum; h, hour; min, minutes; SPEC, specific binding; STR, striatum.
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