Neurite Outgrowth and Gene Expression Profile Correlate with Efficacy of Human Induced Pluripotent Stem Cell-Derived Dopamine Neuron Grafts

Abstract

Transplantation of human induced pluripotent stem cell-derived dopaminergic (iPSC-DA) neurons is a promising therapeutic strategy for Parkinson's disease (PD). To assess optimal cell characteristics and reproducibility, we evaluated the efficacy of iPSC-DA neuron precursors from two individuals with sporadic PD by transplantation into a hemiparkinsonian rat model after differentiation for either 18 (d18) or 25 days (d25). We found similar graft size and dopamine (DA) neuron content in both groups, but only the d18 cells resulted in recovery of motor impairments. In contrast, we report that d25 grafts survived equally as well and produced grafts rich in tyrosine hydroxylase-positive neurons, but were incapable of alleviating any motor deficits. We identified the mechanism of action as the extent of neurite outgrowth into the host brain, with d18 grafts supporting significantly more neurite outgrowth than nonfunctional d25 grafts. RNAseq analysis of the cell preparation suggests that graft efficacy may be enhanced by repression of differentiation-associated genes by REST, defining the optimal predifferentiation state for transplantation. This study demonstrates for the first time that DA neuron grafts can survive well in vivo while completely lacking the capacity to induce recovery from motor dysfunction. In contrast to other recent studies, we demonstrate that neurite outgrowth is the key factor determining graft efficacy and our gene expression profiling revealed characteristics of the cells that may predict their efficacy. These data have implication for the generation of DA neuron grafts for clinical application.

Keywords: Parkinson's disease; behavior; cell therapy; dopamine; gene expression; graft; hiPSC; neural transplantation; neurite outgrowth; rodent model.

FIG. 1.

Experimental Overview. (A) Study design showing experimental progression from fibroblasts to transplant-stage neuronal precursors, and the main outcome measures from both in vitro and in vivo investigation of their suitability as a DA neuron replacement source. (B) Schematics showing unilateral 6-OHDA MFB lesion model with accompanying unilateral TH depletion in immunolabeled brain sections in the STR and SNc (left), and cell replacement strategy, which consists of surgical implantation of cells into the STR (right). 6-OHDA, 6-hydroxydopamine; DA, dopamine; MFB, medial forebrain bundle; STR, striatum; SNc, substantia nigra pars compacta.

FIG. 2.

Characterization of dopaminergic neuron differentiation and maturation from two PD donor iPSC lines. (A) iPSCs from 410 to 411 cell lines that were differentiated to day 80 in vitro express DA neuron marker TH (green). Single-cell patch clamp analysis of day-75 cells showed that most of the neurons had pace making activity and mature membrane resistance (Ra) and capacitance (Cm). Action potential amplitudes were maintained close to 60 mV. HPLC analysis of day 88–89 cultures stimulated with KCl shows release of DA and small amounts of NE and 5-HT. (B) Representative examples of immunohistochemical analysis of TH+ grafts in Control, Lesion, d18 and d25 graft groups. 5-HT, serotonin; HPLC, high performance liquid chromatography; iPSCs, induced pluripotent stem cells; NE, norepinephrine; PD, Parkinson's disease; TH, tyrosine hydroxylase.

FIG. 3.

Effect of iPSC-derived cells on motor function and immunohistochemical analysis of d18 and d25 iPSC-derived grafts. (A) Raw data showing performance of Control, Lesion, 410-d18, 411-d18, 410-d25, and 411-25 grafted rats on amphetamine-induced rotation test across the full test period, and (B) net rotation score at 24 weeks postgraft only. Based on histological analysis of the grafts from d18 and d25 preparations, we present (C) the total mean HuNu+ cells, (D) graft volume, (E) the density of TH+ cells per mm³, (F) total TH+ neurons, (G) percentage of TH+ cells out of total HuNu+ cells, (I) the total GIRK2+ cells, and (J) the percentage of GIRK2+ cells out of TH+ cells. The total AADC+ cell data are depicted in (L) and the percentage of AADC+ cells relative to HuNu+ cells is in (M). Representative immunohistochemistry is presented for (H) TH, (K) GIRK2 (blue) and HuNu (brown), and (N) AADC (brown) and HuNu (blue). P** ≤0.001, error bars = ±SEM. SEM, standard error of the mean.

FIG. 4.

Greater innervation from d18 grafts. (A) Differences were evident in neurite outgrowth from the d18 and 25 grafts into the surrounding host STR up to 1,400 μm from the graft border. (B–E) Representative images of TH+ neurites. (F) Functional recovery did not correlate with graft volume or cellular composition of the grafts. Functional recovery did correlate significantly with greater overall neurite outgrowth and greater fiber projections into both lateral and medial neostriatum (*P ≤ 0.01; **P ≤ 0.001). (G) Manual counting of fibers revealed 4-fold higher average density of projections from d18 grafts compared with d25 grafts. (H) Significantly more neurite outgrowth was also evident in d18 grafts when the tissue was analyzed using unbiased optical density estimates through computerized image analysis (ImageJ). Analysis of the y-intercept of projection trend lines confirmed significantly more neurite outgrowth from d18 grafts (Supplementary Fig. S1C). This is further supported by correlation analysis of both projection density measurements and optical density of projection fibers (Supplementary Fig. S1E–J).

FIG. 5.

Gene expression analysis. RNA was sequenced at the two stages used for efficacy assessment (d18 and d25) and two earlier stages, day 0 (undifferentiated iPSCs) and d13 of differentiation. (A) Principal component one (PC1) explained 56.1% of the variance in transcriptome expression in the d0, d13, d18, and d25 dataset and largely separated the undifferentiated cells (triangles: day 0) from the differentiating dopaminergic precursors (circles, diamonds, and squares representing day 13, 18, and 25, respectively). The second axis (PC2) delineated the DA precursors in a rank order corresponding with their development duration. (B) Comparison of the differentiation stages (d13, d18, d25) showed a strong signal of development stage along PC1 (49.4% of transcriptome variance), and PC2 separated the two cell lines (410: blue and 411: orange) within a time point. (C) Transcription factor binding-site motifs in differentially expressed genes that were enriched in genes upregulated and downregulated between d18 and d25 of differentiation. Genes regulated by the repressors REST (RE1 Silencing Transcription Factor) and SUZ12 (SUZ12 Polycomb Repressive Complex 2 Subunit) were expressed at higher levels at d25. Day-25 cells had lower levels of genes regulated by cell cycle-associated factors FOXM1 and EFF4. (D, E) Normalized, zero-centered gene expression levels of REST and neuronal genes repressed by REST. As cells differentiated, REST decreased and REST targets increased in both iPSC line 410 (D) and iPSC line 411 (E). (F) List of neuron-associated genes with REST motifs upregulated at d25. EFF4, E2F transcription factor 4; FOXM1, Forkhead Box M1.

FIG. 6.

RNAseq analysis of cell preparations. (A) A volcano plot showing that a total of 520 genes were upregulated at day 25 relative to day 18 (pink). Four hundred twenty-nine genes were downregulated between day 18 and 25 (green) (FDR <0.05). (B) RNAseq data for cell preparations harvested at d13, d18, and d25, with genes grouped according to association with cell type or stage. Expression values are displayed as variance stabilized data (vsd), calculated using the vst command in the DESeq2 package [12]. FDR, false discovery rate.