Prolonged Differentiation of Neuron-Astrocyte Co-Cultures Results in Emergence of Dopaminergic Neurons

Abstract

Dopamine is present in a subgroup of neurons that are vital for normal brain functioning. Disruption of the dopaminergic system, e.g., by chemical compounds, contributes to the development of Parkinson's disease and potentially some neurodevelopmental disorders. Current test guidelines for chemical safety assessment do not include specific endpoints for dopamine disruption. Therefore, there is a need for the human-relevant assessment of (developmental) neurotoxicity related to dopamine disruption. The aim of this study was to determine the biological domain related to dopaminergic neurons of a human stem cell-based in vitro test, the human neural progenitor test (hNPT). Neural progenitor cells were differentiated in a neuron-astrocyte co-culture for 70 days, and dopamine-related gene and protein expression was investigated. Expression of genes specific for dopaminergic differentiation and functioning, such as LMX1B, NURR1, TH, SLC6A3, and KCNJ6, were increasing by day 14. From day 42, a network of neurons expressing the catecholamine marker TH and the dopaminergic markers VMAT2 and DAT was present. These results confirm stable gene and protein expression of dopaminergic markers in hNPT. Further characterization and chemical testing are needed to investigate if the model might be relevant in a testing strategy to test the neurotoxicity of the dopaminergic system.

Keywords: dopaminergic neurons; human embryonic stem cells; in vitro; neurodegeneration; neurotoxicity.

Figure 1

Gene expression over the course of neuronal-glial differentiation. (A) Timeline of the culture protocol and time points of sampling. Arrow indicates replating to a new culture plate. (B) Gene expression over time of a selection of dopaminergic markers for cell types relative to the neural progenitor cell (NPC) culture, ordered by expected timing of expression: early markers for differentiation of dopaminergic progenitor cells (LMX1B), differentiation of dopaminergic neurons (NURR1, PITX3), a catecholaminergic neuron marker (TH) and proteins that can transport dopamine (SLC618A2, SLC6A3) or are involved in dopaminergic neurotransmission (KCNJ6). (C) Gene expression over time of a selection of markers for cell types relative to the NPC culture: NPC (NEUROG1, NES), neurons (TUBB3, MAP2), astrocytes (GFAP), synapses (SYNPR, DLG4), excitatory (SLC17A6) and inhibitory (SLC32A1) neurotransmitter vesicles. NP: neural progenitor medium, ND: neural differentiation medium CNTF: Ciliary neurotrophic factor. Significance (adjusted p-value ≤ 0.05) is indicated relative to *: NPC, ^: 28 days, ~: 42 days, #: 56 days, +: 63 days. Full statistics can be found in Supplementary Table S1.

Figure 2

Differentiation of NPC to neuron-astrocyte culture including dopaminergic neurons. (A) Timeline of the culture protocol and time points of sampling. Arrow indicates replating to a new culture plate. (B) Light images of the neuronal culture differentiating over time. (C) Maximum projections (5 μm) of the same time points as in (B) showing neurons (TAU, MAP2) and TH⁺ neurons. Scale bars: (B)—200 μm, (C)—20 μm.

Figure 3

Differentiating NPCs express specific markers for dopaminergic neurons. (A–D) Maximum projections (5 μm) of the same time points as in Figure 1 showing neurons that express TH and transporter proteins VMAT2 and DAT. (A’–D’) XZ projection of (A–D). Yellow dot in corresponding image indicates where slice was made. (E) Maximum projections (10 μm) and single images showing intense staining of VMAT2 in GFAP⁺ astrocytes. Scale bar: 20 μm.