Human Pluripotent Stem Cell-Derived Medium Spiny Neuron-like Cells Exhibit Gene Desensitization

Abstract

Gene desensitization in response to a repeated stimulus is a complex phenotype important across homeostatic and disease processes, including addiction, learning, and memory. These complex phenotypes are being characterized and connected to important physiologically relevant functions in rodent systems but are difficult to capture in human models where even acute responses to important neurotransmitters are understudied. Here through transcriptomic analysis, we map the dynamic responses of human stem cell-derived medium spiny neuron-like cells (hMSN-like cells) to dopamine. Furthermore, we show that these human neurons can reflect and capture cellular desensitization to chronic versus acute administration of dopamine. These human cells are further able to capture complex receptor crosstalk in response to the pharmacological perturbations of distinct dopamine receptor subtypes. This study demonstrates the potential utility and remaining challenges of using human stem cell-derived neurons to capture and study the complex dynamic mechanisms of the brain.

Keywords: RNA-seq; acute and chronic; dopamine; gene desensitization; human embryonic stem cell; medium spiny neuron.

Figure 1

hPSC-derived neurons express markers of MSNs. (A) hPSCs are differentiated into hMSN-like cells following an Activin A induction protocol (Arber et al., 2015 [17]), (B) immunostained for MAP2+ (red), DARPP32 (green), and DAPI (blue) and quantified for DARPP32+ percentage. GFP-transfected hMSN-like cells were imaged to better highlight cell morphology. Scale bar = 50 µm. (C) QRT-PCR of RNA isolated from H1 (blue) and H9 (orange) hPSCs and hPSCs differentiated into hMSN-like cells at DIV 16 (D16) and DIV 45 (D45), for genes of interest. Values were normalized to HPRT1 mRNA levels in the same samples and expressed as normalized fold changes in hMSN-like versus hPSC cells. Values normalized to GUSB are provided in Figure S1. Gene categories are labeled in red. n = 3–4 independent replicates & 2 technical replicates. Error bars = Standard error.

Figure 2

hMSN-like cells exhibit dose and time-dependent responses to dopamine. (A) QRT-PCR of RNA isolated from DIV45 H1 and H9 hMSN-like cells 1 h after exposure to different dopamine concentrations (1 µM to 10 mM) and analyzed for FOSB and FOS. (B) QRT-PCR of RNA isolated from DIV 45 H1 and H9 hMSN-like cells 0 to 120 min after exposure to 1 mM dopamine and analyzed for FOSB and FOS. (A,B) For QRT-PCR, values were normalized to GUSB mRNA levels in the same samples and expressed as a fold change in dopamine versus PBS control cultures. * = p < 0.05; One-way ANOVA. n = 3–4 independent replicates and 2 technical replicates. (C) Venn Diagram showing the number of shared differentially expressed genes (DEG) between DIV45 hMSN-like cells quantified by RNA-seq 1 hour after acute 1 μM and 1 mM dopamine. (D) Functional enrichment analysis of RNA-seq data for KEGG pathways of DEGs unique to DIV45 1 μM dopamine dosed (left) and 1 mM dosed (right) hMSN-like cells. Significance is represented by Log₁₀-transformed p-values. Dotted red line indicates p-value of 0.05. (C,D) DEGs were identified by max group mean ≥ 0.75, FDR p-value < 0.05, and Log₂(Fold Change) > |1|. Differential expression was performed against PBS control group using the Wald test. n = 3 independent replicates.

Figure 3

Chronic administration of dopamine leads to desensitization of genes implicated in cocaine and dopamine responses. (A) Schematic for isolation of RNA from H9 hMSN-like cells dosed acutely (DIV45) and chronically (DIV50) with dopamine. (B) Total number of DEGs from RNA-seq of H9 hMSN-like cells dosed with 1 μM and 1 mM dopamine. (C) Venn diagrams showing shared number of DEGs 1 hour after dosage between hMSN-like cells dosed with DIV45 acute and DIV50 chronic dopamine. (D) Heatmaps of top 20 desensitized genes for hMSN-like cells exposed to acute and chronic dopamine. Desensitized genes are defined as the ratio of Acute 1 h Log₂(Fold Change)/Chronic 1 h Log₂(Fold Change) > |1.1|. Overlapping genes highlighted by tan-colored bars. (E) IPA upstream regulators and gProfiler transcription factor regulatory motifs of desensitized genes common between 1 μM and 1 mM dopamine conditions. (F) Gene ontology biological processes for highly desensitized genes after chronic 1 mM dopamine, defined when the ratio Acute 1 h Log₂(Fold Change)/Chronic 1 h Log₂(Fold Change) > |2|. (E,F) Significance is represented by Log₁₀-transformed p-values with dotted red line indicating p-value of 0.05. In all cases, data were obtained from RNA-seq of H9 hMSN-like cells. DEGs were identified by max group mean ≥ 0.75, FDR p-value < 0.05, and Log₂(Fold Change) > |1|. Differential expression was performed against PBS control group using the Wald test. n = 3 independent replicates.

Figure 4

Time course of chronic dopamine administration reveals peak in DEGs at day 3 and desensitization at day 5. (A) Total number of DEGs from RNA-seq of DIV50 hMSN-like cells 1 h after 2, 3, 4, or 5 days of daily dosing of 1 mM dopamine. (B) Venn diagrams showing shared (white) and unique (red) numbers of DEGs between hMSN-like cells dosed with dopamine for 2–5 days. (C) Gene ontology biological processes and KEGG pathways for shared and unique genes from hMSN-like cells dosed with dopamine for 2–5 days. Significance is represented by Log₁₀-transformed p-values with dotted red line indicating p-value of 0.05. In all cases, data were obtained from RNA-seq of H9 hMSN-like cells. DEGs were identified by FDR p-value < 0.05. Differential expression was performed against ascorbic acid vehicle control group using the Wald test. n = 2–3 independent replicates.

Figure 5

hMSN-like cells capture some features of dopamine receptor cross-interactions. (A) Total number of DEGs from RNA-seq of DIV45 hMSN-like cells dosed with receptor agonists. (B) Gene ontology molecular functions and reactome pathways for DEGs from hMSN-like cells dosed with ADORA2A agonist CGS21680 and D2-like receptor agonist quinpirole. Dotted red line indicates p-value of 0.05. (C) Venn diagram showing shared and unique numbers of DEGs for DIV45 hMSN-like cells dosed with agonists. In all cases, data were obtained from RNA-seq of DIV45 H9 hMSN-like cells. DEGs were identified by FDR p-value < 0.05. Differential expression was performed against a water (vehicle) control group using the Wald test. n = 3 independent replicates.