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
View: Text | PDF
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

×

Figure 4

Characterization of grafts and behavioral analysis.

Options: View larger image (or click on image) Download as PowerPoint
Characterization of grafts and behavioral analysis. (A–F) Immuno­histo­c...
(A–F) Immuno­histo­chemical analysis of GFP, TH, and DAPI expression 6 weeks after grafting (examples from single-sorted grafts). (G–I) Immuno­histochemical analyses of GFP, FOXA2, and TH expression. Counts of TH+ cells in the grafts plotted against total volume in (J) single-sorted and (K) double-sorted cell grafts. Analysis of ipsilateral rotations at stages before and after grafting in mice grafted with (L) single-sorted cells (n = 4 for each line) and (M) double-sorted cells (n = 3 for each line), including sham-based (n = 4) and J1 nontransgenic (n = 3) controls. Scale bar: 200 μm (A–C); 50 μm (D–I). *P ≤ 0.05, Dunnett’s test with Hes5 grafts as baseline group for J and Pitx3 grafts as baseline group for K, L, and M. *P ≤ 0.0, **P ≤ 0.005, ANOVA analysis.