Dopaminergic neurons from midbrain-specified human embryonic stem cell-derived neural stem cells engrafted in a monkey model of Parkinson's disease

Abstract

The use of human embryonic stem cells (hESCs) to repair diseased or injured brain is promising technology with significant humanitarian, societal and economic impact. Parkinson's disease (PD) is a neurological disorder characterized by the loss of midbrain dopaminergic (DA) neurons. The generation of this cell type will fulfill a currently unmet therapeutic need. We report on the isolation and perpetuation of a midbrain-specified self-renewable human neural stem cell line (hNSCs) from hESCs. These hNSCs grew as a monolayer and uniformly expressed the neural precursor markers nestin, vimentin and a radial glial phenotype. We describe a process to direct the differentiation of these hNSCs towards the DA lineage. Glial conditioned media acted synergistically with fibroblastic growth factor and leukemia inhibitory factor to induce the expression of the DA marker, tyrosine hydroxylase (TH), in the hNSC progeny. The glial-derived neurotrophic factor did not fully mimic the effects of conditioned media. The hNSCs expressed the midbrain-specific transcription factors Nurr1 and Pitx3. The inductive effects did not modify the level of the glutamic acid decarboxylase (GAD) transcript, a marker for GABAergic neurons, while the TH transcript increased 10-fold. Immunocytochemical analysis demonstrated that the TH-expressing cells did not co-localize with GAD. The transplantation of these DA-induced hNSCs into the non-human primate MPTP model of PD demonstrated that the cells maintain their DA-induced phenotype, extend neurite outgrowths and express synaptic markers.

Figure 1. The hNSCs uniformly express markers of neural stem cells.

The isolated hNSCs were passaged every 5–7 days, as described in the Methods section. They grew as a stable homogenous monolayer and uniformly expressed molecular features of NSCs. The top 3 panels: A, B, C represent photomicrographs of undifferentiated hNSCs immunostained with the neural precursor markers nestin (red, A) and vimentin (green, B) and the radial glial marker 3CB2 (purple, C). The cultures were counterstained with the nuclear DNA marker 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, blue). The bottom panels (D, E, F) show 7-day old differentiated hNSC cultures processed for indirect immunocytochemistry. Differentiated hNSCs expressed the neuronal-specific marker TuJ1 (green D, E), the astroglia marker GFAP (red, E), the oligodendroglial marker GC (green, F) and DAPI, blue nuclei in all panels. Cell nuclei were stained with live cell marker DAPI (blue, F, G). Scale bars are 20 µm (A–F).

Figure 2. Treatment with the glial conditioned media (CM) and growth factors (GF) induce TH expression in hNSCs derived from hESCs.

hNSCs were single cell dissociated in a defined medium and plated on poly-L-ornithine-coated glass coverslips either under control conditions (A) or in glial cell line conditioned media supplemented with the growth factors bFGF (20 ng/ml) and LIF 10 ng/ml for 10 days (B & C). Fixed cultures were stained with nuclear marker DAPI (blue) and processed for TH (red) and for the neuronal marker TuJ1 (green) immunocytochemistry, as described in Materials and Methods. Scale bars are 50 µm (A–C).

Figure 3. Electrophysiological properties of DA induced neurons.

Quantitative analysis of the TH-inductive effects (A & B). The TH-IR neurons were expressed as a percentage of total live cells determined by DAPI staining and also as a percentage of the total number of TuJ1-IR neurons. Results are mean ± S.E.M. of experiments performed three times on independent culture preparations, each performed in duplicate. The TH induction with CM+F+L treatment produced significantly more TH+ cells compared to CM treatment alone (*P<0.01) and to non-treated control (**P<0.001). (C) The relative abundance of TH and 18 S transcripts was assessed by RT-PCR (see Materials and Methods section for the description of the primers used and the PCR conditions) in control (Ctr), GDNF+bFGF (F)+LIF (L) and in CM+F+L treated cultures (line +) which showed a strong induction of TH in CM+F+L culture condition. (D) Representative current clamp trace of action potentials elicited in response to a 400 pA current injection from a neuron. (E) Example of action potential burst in response to 400 pA current injections. (F) Voltage-activated sodium and potassium currents elicited in response to a −50 mV step. (G) Sample trace of spontaneous EPSCs in a neuron held at −70 mV. (H) Triple labeling immunocytochemical process of hNSC culture used for electrophysiological recording showing the co-localization of TH with biocytin and DAT. Scale bar is 20 µm (H).

Figure 4. CM + GF stimulate TH and midbrain gene expression but not GAD.

(A): Total RNA samples were extracted from hNSCs cultured for 10 days in the absence (line −) or presence (line +) of CM + bFGF + LIF. The relative abundance of TH, Nurr1, Pitx3, GAD and 18 S was assessed by RT-PCR (see Materials Methods section for the description of the primers used and the PCR conditions). CM+GF treated cultures (line +) showed a selective increase in PAX2, En1, TH, and DDC transcripts. (B, C): Gel densitometry software (Image J) was used to quantify amplified PCR products in 3 independent experiments. TH mRNA was significantly increased in CM+GF treated culture in comparison to control (* p<0.01), while GAD mRNA expression did not show significant differences between control and CM+GF treated cultures (ns  =  not significant). CM+GF treated cultures were immunostained for TH/Pitx3/Nurr1 (D) and TH/FoxA2/DAT (E). Photomicrographs in (F) show example of distinct cells expressing either the GABAergic maker GAD (purple, arrow head) or the DA marker TH (red, arrow). Scale bars are 50 µm (D), 20 µm (E), 10 µm (F).

Figure 5. Grafted dopamine-induced hNSCs maintain their phenotype in MPTP-lesioned NHP.

Coronal section through the caudate/putamen of an MPTP-lesioned NHP (A), show transplant (TX) of hNSCs counterstained with the nuclear marker DAPI (blue) and immunoprocessed for the human-specific cytoplasmic (purple, hCyt) and the TH (red). (B) Photomicrograph taken from the edge of the graft showing triple labeling with hCyt (purple), TH (red) and the synaptic marker synaptophysin (green). An example of TH+ cell is shown in C, and photomicrograph in D show that the neuritic process extended by the hCyt+ grafted cells (purple) do not express the serotoninergic marker (5 HT, green) indicated by the arrow and expressed by endogenous neurons. The TH+ neurite outgrowth express synaptophysin+ puncta (E). Scale bars are 200 µm (A), 20 µm (B–C), 10 µm (D–E).

Figure 6. Human NSCs engraft, extend neurites, and differentiate into TH+ neurons in MPTP-lesioned NHPs.

Coronal section through the midbain showing grafted DA-differentiated hNSCs into the substantia nigra. Two months-post-transplantation, brains were perfused, sectioned, & immunostained for human-specific & TH markers. Arrows indicate two examples of engrafted donor-derived cells, previously transfected with lenti-GFP, coexpressing the nuclear marker DAPI (bleu), the human nuclear marker hNuc (purple), GFP (green) & TH (red). Scale bar is 50 µm.