Immature Midbrain Dopaminergic Neurons Derived from Floor-Plate Method Improve Cell Transplantation Therapy Efficacy for Parkinson's Disease

Abstract

Recent reports have indicated human embryonic stem cells-derived midbrain dopamine (mDA) neurons as proper cell resources for use in Parkinson's disease (PD) therapy. Nevertheless, no detailed and systematic study has been conducted to identify which differentiation stages of mDA cells are most suitable for transplantation in PD therapy. Here, we transplanted three types of mDA cells, DA progenitors (differentiated in vitro for 16 days [D16]), immature DA neurons (D25), and DA neurons (D35), into PD mice and found that all three types of cells showed high viability and strong neuronal differentiation in vivo. Both D25 and D35 cells showed neuronal maturation and differentiation toward TH⁺ cells and, accordingly, satisfactory behavioral functional recovery. However, transplanted D16 cells were less capable of producing functional recovery. These findings provide a valuable guideline for standardizing the differentiation stage of the transplantable cells used in clinical cell therapy for PD. Stem Cells Translational Medicine 2017;6:1803-1814.

Keywords: Differentiation; Floor-plate; Midbrain dopaminergic neurons; Parkinson's disease; Transplantation; Tyrosine hydroxylase.

Figure 1

Differentiation and characterization of hESC‐derived mDA neurons. (A): An overview of the floor plate (FP)‐based mDA neuron differentiation protocol and stages for transplantation. (B): Flow cytometric analysis and, (C): and (D): quantitative RT‐PCR analysis of gene expression levels at different stages of differentiation. Data are shown as the mean ± SD, n = 3 (independent experiments). (E): Morphology of mDA cells at different stages of differentiation. Scale bar: 50 μm. (F): Immunofluorescence images of various stages of cell aggregates on day 16 (OTX2/LMX1A), day 25 (TH/NURR1), and day 35 (TH/MAP2). Scale bar: 50 μm. (G): Quantification of TH⁺ and Tuj1⁺ cell populations on day 25 and day 35 by flow cytometry. (H): Action potential induced by 50 pA current recorded from hESC‐derived neurons on day 42. (I): Spontaneous postsynaptic current at Vm = −60 mV recorded from hESC‐derived neurons on day 42. (J): HPLC analysis of dopamine and its metabolite, DOPAC, from differentiated cells on day 51. Abbreviations: hESC, human embryonic stem cell; mDA, midbrain dopaminergic.

Figure 2

The survival and proliferation of the transplanted cells 3 months post‐transplantation. (A): IHC‐DAB staining using human‐specific NCAM (hNCAM) antibody show the graft sizes of transplantations of the three stages of mDA cells. Red dotted lines outline coronal sections of the right cortical hemisphere. Scale bar: 600 µm. (B): Representative images showing the survival and proliferation of three stages of mDA cells. hNUC staining revealing the surviving transplanted cells. Ki67 staining showed the proliferating cells among the transplanted cells. Scale bar: 100 µm. (C): Bar chart showing the quantification of the surviving transplanted cells in the three types of transplants. Data are shown as the means ± SD, n = 3 animals, Student's t test (two‐tailed), *p < .05. (D): Bar chart showing the percentage of proliferating cells among the grafted cells in the three types of transplants. Data are shown as the means ± SD, n = 3 animals, Student's t test (two‐tailed). Abbreviations: IHC, Immunohistochemistry; DAB, 3,3′‐diaminobenzidine; mDA, midbrain dopaminergic; hNUC, Human specific nuclear antigen.

Figure 3

Neuronal differentiation and maturation of three types of transplants. (A): The majority of the transplanted cells (labeled with hNCAM, red) differentiated into neurons (labeled with TUJ1, green). Scale bars: 50 µm in enlarged panel, 200 µm in other panels. (B): Representative images showing the mature neurons (labeled with NEUN, green) derived from the transplanted D16, D25, and D35 mDA cells (labeled with hNUC, red) 3 months post‐transplantation. G: grafted cells; H: host tissue. Scale bar: 50 µm. (C): Bar chart showing the percentage of the mature neurons among the surviving transplanted cells in the three types of transplants. Data are shown as the means ± SD, n = 3 animals, Student's t test (two‐tailed). Abbreviations: mDA, midbrain dopaminergic; hNUC, Human specific nuclear antigen.

Figure 4

Comparison of the functional efficacy of the three types of transplants. (A): Subsets of the transplanted cells (hNUC positive, red) could differentiate into TH⁺ DA neurons (green). G: graft region; H: host tissue. Scale bar: 50 µm in enlarged images, 100 μm in the other images. (B): Bar chart showing the percentage of mDA neurons among the surviving transplanted cells for the three types of transplants. Data are shown as the means ± SD, n = 3 animals, Student's t test (two‐tailed). (C): Immunofluorescence images showing the co‐expression of TH with midbrain neuronal markers, LMX1A, FOXA2, and NURR1, in the grafted D25 mDA neurons. 4′,6‐iamidino‐2‐phenylindole (DAPI) (blue) indicates cell nuclei. White arrowheads indicate the cell bodies of double‐positive cells. Scale bar: 50 µm. (D): Immunofluorescence images showing the co‐expression of TH with A9 subtype marker, GIRK2, in the grafted D25 mDA neurons. DAPI (blue) indicates cell nuclei. White arrowheads indicate the cell bodies of double‐positive cells. Scale bar: 50 µm. (E): Some of the transplanted cells (hNUC positive, red) could differentiate into GABAergic neurons (green). White arrowheads indicate GABAergic neurons. Scale bar: 50 µm. (F): Bar chart showing the efficacy of behavioral recovery produced by the three types of transplants. Amphetamine‐induced ipsilateral rotations recorded 3 months post‐transplantation were compared with rotations recorded pre‐transplantation. Data are presented as percentages normalized to the pre‐transplantation recordings. The values represented means ± SD, n = 5 animals, Student's t test (two‐tailed), *p < .05, **p < .01. Abbreviations: mDA, midbrain dopaminergic; hNUC, Human specific nuclear antigen.

Figure 5

The transplanted D25 mDA neurons innervated multiple brain regions. IHC‐DAB staining using hNCAM antibody revealed the projections of the transplanted human ES‐derived D25 mDA neurons. (A): Sagittal section showed that the transplanted cells residing in the transplanted core (T) innervated the surrounding striatum (B) and projected toward the pons and cerebellum through the cerebral peduncle (C). (D): Coronal section showed that the transplanted cells residing in the transplanted core innervated the septal nuclei (E), the ipsilateralpiriform cortex (F), the contralateral piriform cortex (G), and the surrounding striatum (H). (I): The migration of transplanted cells from the transplantation core to the surrounding striatum. The thick dotted line outlined the edge of the transplantation core region. The thin dotted line indicates the frontier of the migrated transplanted cells. White arrows indicate host cells present in the transplantation core region. T: transplanted core; Hi: hippocampus; CC: corpus callosum. Scale bars: 500 µm in (A) and (D); 50 µm in (B), (C), (E–H); 100 µm in (I). Abbreviations: IHC, immunohistochemistry; DAB, 3,3′‐diaminobenzidine; mDA, midbrain dopaminergic.