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.

Figure 1. Characterization of reporter expression in the 3 BAC lines.

(A–C) Live reporter expression on day 14 of differentiation. (D–F) Overlay of reporter expression and phase-contrast images. (G–I) FACS analysis for reporter expression at day 14 of differentiation plotted against PE and FITC channels. Numbers represent the percentage of positive cells. Scale bar: 50 μm.

Figure 2. Short-term characterization of marker expression in FACS-purified cells.

(A) qRT-PCR data showing enrichment of Nurr1 (14.6 fold) and Pitx3 (10.0 fold) in reporter⁺ cells compared with that in reporter^– cells of the respective cell lines. (B–K) Immuno­cyto­chemical analysis of reporter⁺ cells 24 hours after sorting. (B) Quantitative analysis in Hes5, Nurr1, and Pitx3 lines for coexpression of GFP and TH (GFP⁺/TH⁺, TH⁺/GFP⁺), the percentages of GFP⁺, Tuj1⁺, and TH⁺ cells among total cells (GFP⁺/DAPI⁺, Tuj1⁺/DAPI⁺, TH⁺/DAPI⁺), and the colocalization of midbrain markers EN1, LMX1A, and FOXA2 in TH⁺ cells (EN1⁺/TH⁺, LMX1A⁺/TH⁺, FOXA2⁺/TH⁺). (C–K) Representative images of immuno­cyto­chemical marker analysis. Scale bar: 50 μm.

Figure 3. Long-term in vitro characterization of cells after sorting.

(A–C) Immunocytochemical analysis for GFP, GFAP, and DAPI in the 3 lines. (D–F) Immunocytochemical analysis for GFP, TH, and Tuj1. (G) Phase-contrast and GFP expression in Nurr1::GFP⁺-sorted cells 6 days after replating. (H) Whole-cell recordings in current clamp mode from Nurr1 and Pitx3 lines show neuronal-like excitability, with cells firing multiple action potentials at rates between 2 and 10 Hz and evidence of spike frequency adaptation (“accommodation”). Scale bar: 100 μm (A–F); 40 μm (G).

Figure 4. Characterization of grafts and behavioral analysis.

(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.

Figure 5. Microarray analysis of FACS-purified cell populations.

(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.

Figure 6. In vivo confirmation of UNCX4.1 and PBX1 expression in the adult mouse SN and human fetal midbrain (6–8 weeks after conception).

Immuno­histo­chemistry for UNCX4.1 ([A] UNCX4.1 in single channel; [B] UNCX4.1/TH/DAPI in merged image) and PBX1 ([C] PBX1 in single channel; [D] PBX1/TH/DAPI in merged image) in the adult mouse SN. Immuno­histochemistry in human fetal midbrain tissue for (E–G) UNCX4.1 and (H–J) PBX1 demonstrated similar perinuclear and nuclear staining patterns, respectively. Scale bar: 200 μm (A–D); 100 μm (E, F, H, and I); 25 μm (G, J, and insets for A–D).