Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells

Abstract

We developed a method for the efficient generation of functional dopaminergic (DA) neurons from human embryonic stem cells (hESCs) on a large scale. The most unique feature of this method is the generation of homogeneous spherical neural masses (SNMs) from the hESC-derived neural precursors. These SNMs provide several advantages: (i) they can be passaged for a long time without losing their differentiation capability into DA neurons; (ii) they can be coaxed into DA neurons at much higher efficiency than that from previous reports (86% tyrosine hydroxylase-positive neurons/total neurons); (iii) the induction of DA neurons from SNMs only takes 14 days; and (iv) no feeder cells are required during differentiation. These advantages allowed us to obtain a large number of DA neurons within a short time period and minimized potential contamination of unwanted cells or pathogens coming from the feeder layer. The highly efficient differentiation may not only enhance the efficacy of the cell therapy but also reduce the potential tumor formation from the undifferentiated residual hESCs. In line with this effect, we have never observed any tumor formation from the transplanted animals used in our study. When grafted into a parkinsonian rat model, the hESC-derived DA neurons elicited clear behavioral recovery in three behavioral tests. In summary, our study paves the way for the large-scale generation of purer and functional DA neurons for future clinical applications.

Fig. 1.

Differentiation of hESCs into TH⁺ neurons. Schematic procedures for the in vitro differentiation of hESCs. NPs with neural rosettes (C) and neural-tube-like structures (D) were produced by neural selection and expansion procedures after EB formation (B). An SNM (E) was then made by using these NPs in a suspension culture (stage 1). After four passages, the SNMs were differentiated into TH⁺ neurons by treating the cells with signaling molecules, such as SHH, FGF8, and AA (stage 2). The detection of TH⁺ (I) and βIII-tubulin⁺ cells (J) from differentiated hESCs was performed by immunocytochemistry.

Fig. 2.

Quantitative analysis of TH⁺ and βIII-tubulin⁺ neurons and the differentiation of TH⁺ neurons from other hESC lines and highly passaged SNMs. (A) The proportion of TH⁺ neurons to the total number of neurons. TH⁺ and βIII-tubulin⁺ neurons (≈15,000 cells from three independent experiments) were counted after performing immunocytochemistry. The value represents the mean ± SEM. (B and C) Flow cytometric analysis of TH⁺ and βIII-tubulin⁺ neurons. Anti-βIII-tubulin and anti-TH antibodies were used for the detection of total neurons and TH⁺ neurons. Stained cells were analyzed by using a FACScan. This analysis revealed that ≈85% (65.3%/77.1% = 84.7%) of the total neurons were TH⁺ neurons, and ≈77% of all of the cells were neurons (B, sample analysis; C, control without primary antibody treatment). (D–L) Detection of βIII-tubulin⁺ (green; D, G, and J) and TH⁺ (red; E, H, and K) cells after hESC differentiation. SNUhES3 (passage four; D–F), SNUhES16 (passage four; G–I), and SNUhES1 (highly passaged SNM, passage 10; J–L) were used. (M) TH⁺ and βIII-tubulin⁺ neurons (≈5,000 cells from five independent samples) that were derived from the SNUhES3 and SNUhES16 cell lines were counted after performing immunocytochemistry. The value represents the mean ± SEM.

Fig. 3.

The majority of TH⁺ neurons coexpress other markers specific for midbrain DA neurons. The majority of TH⁺ neurons (A, D, and M) coexpress midbrain DA markers such as AADC (B) and En1 (E) but not GABA (N). Approximately 4% of TH⁺ (G and J) cells expressed noradrenergic or adrenergic markers such as DBH (4.18 ± 0.50%; H) and PNMT (4.44 ± 0.57%; K). ES cell-derived neurons were stained with anti-TH (A, D, G, J, and M), anti-AADC (DA, serotonergic; B), anti-En1 (DA; E), anti-GABA (GABAergic; N), anti-DBH (noradrenergic and adrenergic; H), and anti-PNMT (adrenergic; K) antibodies. (P) Semiquantitative RT-PCR analysis for neural and DA markers during in vitro differentiation of hESCs. The expression levels of each gene were normalized to GAPDH gene expression level. ES, undifferentiated ES cells; EB, embryoid bodies; SNM, spherical neural mass; DA, DA neurons.

Fig. 4.

The hESC-derived DA neurons are biologically functional. (A and B) Electrophysiological properties of DA neurons differentiated from hESCs. (A) Current-clamp recordings during prolonged depolarizing current injections. Bottom traces represent current injections, whereas top traces indicate voltage recordings. The depolarizing current injections elicited fast action potentials. (B) Voltage-dependent membrane currents. Depolarizing voltage steps (bottom traces) elicited outward K⁺ currents and fast inward Na⁺ currents. (C–E) TH⁺ cells (C) coexpress synaptophysin (D), which is a protein necessary for synapse formation. Immunostaining was performed by using antibodies against TH and synaptophysin. (F) Analyses of dopamine release. DA analysis was performed as described in ref. . Results show the HPLC analyses of DA levels in control media (control), 24 h-conditioned media (CM), and 50 mM KCl-challenged media (KM; 30 min) after differentiation.

Fig. 5.

Behavioral recovery by hESC-derived neurons in a parkinsonian rat model. The behavior of PD animals grafted with neurons differentiated from SNUhES1 and sham controls was tested before transplantation (Pre) and at 4, 8, and 12 weeks after the grafting. (A) Apomorphine-induced rotation response per hour (three independent experiments until 12 weeks, n = 6). (B) Amphetamine-induced rotation response per hour (three independent experiments until 12 weeks, n = 6). Only rats that showed a significant turning behavior (>310 turns per hour) after drug administration were used in our study. The rotational scores (rotation %) in A and B are expressed as a percentage of rotations compared with the values obtained from the Pre time point. *, significantly different from sham controls at P < 0.01; **, P < 0.001. (C) The adjusting step test is expressed as the number of steps per 0.9 m on a treadmill (at a rate of 0.075 m/s) with the disabled forepaw (four independent experiments until 12 weeks, n = 14).

Fig. 6.

Characterization and survival of the transplanted cells in a parkinsonian rat model. (A) Tiled coronal sections of the rat brain showing the 6-OHDA-treated side (Left) and the cells in the injection area (human nuclei, green). The normal striatum was stained with anti-TH antibody (red). (B) The human cells (human mitochondria, green) are predominantly βIII-tubulin⁺ (red), indicating that most of the human cells are neurons. (C) Twelve weeks after transplantation, survival of TH⁺ cells was analyzed by immunohistochemistry using anti-human nuclei antibody (green) and anti-TH antibody (red). The TH⁺ cells colocalized with human marker. (D) Ki67⁺ cells (green) were found in the grafted region, indicating the existence of proliferating cells. (E) Oct4⁺ cells were not detected. (F) Association of grafted cells and astrocytes was confirmed by immunostaining using anti-human nuclei (green) and anti-GFAP (red) antibodies. (G) Immunostaining of DAT (red) to confirm the maturity of the grafted DA neurons; human nuclei (green).