Identification of embryonic stem cell–derived midbrain dopaminergic neurons for engraftment
Yosif M. Ganat, Elizabeth L. Calder, Sonja Kriks, Jenny Nelander, Edmund Y. Tu, Fan Jia, Daniela Battista, Neil Harrison, Malin Parmar, Mark J. Tomishima, Urs Rutishauser, Lorenz Studer
Yosif M. Ganat, Elizabeth L. Calder, Sonja Kriks, Jenny Nelander, Edmund Y. Tu, Fan Jia, Daniela Battista, Neil Harrison, Malin Parmar, Mark J. Tomishima, Urs Rutishauser, Lorenz Studer
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Research Article

Identification of embryonic stem cell–derived midbrain dopaminergic neurons for engraftment

Abstract

Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurr1+ stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery.

Authors

Yosif M. Ganat, Elizabeth L. Calder, Sonja Kriks, Jenny Nelander, Edmund Y. Tu, Fan Jia, Daniela Battista, Neil Harrison, Malin Parmar, Mark J. Tomishima, Urs Rutishauser, Lorenz Studer

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Figure 5

Microarray analysis of FACS-purified cell populations.

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Microarray analysis of FACS-purified cell populations. (A) Venn diagram ...
(A) Venn diagram plot for significantly enriched (increased expression only) genes in intraline analysis, showing 107 genes common to both Nurr1::GFP+ and Pitx3::YFP+. (B) Venn diagram plot of significantly enriched genes in interline analysis, showing 148 genes common to both Nurr1::GFP+ and Pitx3::YFP+ cells in contrast to Hes5::GFP+ cells. (C–E) Scatter plots of significantly altered genes (≥2 fold increased/decreased; P ≤ 0.05). Red boxes represent genes enriched at the Hes5 stage (H+/H–), while blue boxes represent genes that are decreased at that stage. (C) Intraline scatter plot of the 233 altered H+/H– genes (x axis) compared with the altered 232 N+/N– genes (y axis) (449 genes common to both). (D) Intraline scatter plot of the 232 altered N+/N– genes compared with the 556 altered P+/P– genes (656 genes common to both). (E) Interline scatter plot of the 586 altered N+/H+ genes compared with the 1,203 altered P+/H+ genes (1,355 genes common to both). (F) Dendrogram plot of all significantly altered genes (P ≤ 0.05) in all 3 reporter+ and reporter– sorts across the 3 lines. H, Hes5; P, Pitx3; N, Nurr1.