Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs

Abstract

Generation of functional dopamine (DA) neurons is an essential step for the development of effective cell therapy for Parkinson's disease (PD). The generation of DA neurons can be accomplished by overexpression of DA-inducible genes using virus- or DNA-based gene delivery methods. However, these gene delivery methods often cause chromosomal anomalies. In contrast, mRNA-based gene delivery avoids this problem and therefore is considered safe to use in the development of cell-based therapy. Thus, we used mRNA-based gene delivery method to generate safe DA neurons. In this study, we generated transformation-free DA neurons by transfection of mRNA encoding DA-inducible genes Nurr1 and FoxA2. The delivery of mRNA encoding dopaminergic fate inducing genes proved sufficient to induce naive rat forebrain precursor cells to differentiate into neurons exhibiting the biochemical, electrophysiological, and functional properties of DA neurons in vitro. Additionally, the generation efficiency of DA neurons was improved by the addition of small molecules, db-cAMP, and the adjustment of transfection timing. The successful generation of DA neurons using an mRNA-based method offers the possibility of developing clinical-grade cell sources for neuronal cell replacement treatment for PD.

Keywords: Parkinson’s disease; dopamine neuron; genomic integration free; in vitro transcription; mRNA; neural precursor cell.

Graphical abstract

Figure 1

Protein Expression by Transfection of In Vitro-Synthesized mRNAs (A) Schematic diagrams of the DNA templates for mRNA synthesis. To induce protein expression of the genes of interest (eGFP, FLAG-tagged Nurr1, and HA-tagged FoxA2), plasmid pcDNA3.1(+) was modified by insertion of sequences containing a 5′UTR, 3′UTR, and poly A tail (120 pA). (B) Protein expression of synthetic mRNA. The synthetic mRNAs were transfected into HEK293 cells and rat NPCs using liposomal transfection (TF) reagents. Proteins translated from each mRNA were stained with corresponding antibodies (anti-NURR1 or anti-HA) at day 1 after transfection, and fluorescent microscopic images were shown. The histograms on the right show that treatment with db-cAMP increased protein expression from in vitro synthesized mRNAs. Error bars represent SE from the results of three independent experiments. Paired t test was used to assess differences between two groups (***p < 0.001). The scale bar represents 20 μm.

Figure 2

Nurr1 mRNA Transfection Induces TH Expression in Rat Embryonic Cortical Neural Precursor Cells (A) Protein expression by repetitive mRNA transfection (TF). The synthetic eGFP mRNA or Nurr1 mRNA was repetitively transfected into rat NPCs once per day for 10 days. Transfected cells were fixed on days 1, 3, 7, and 10 and were stained with anti-TH antibody. (B and C) Positive cells (GFP or TH) were scored on days 1, 3, and 7. Error bars represent SE from the results of three independent experiments. Paired t test was used to assess differences between two groups (*p < 0.05, **p < 0.01, ***p < 0.001). The scale bar represents 20 μm.

Figure 3

Increased Differentiation to DA Neurons by Additional Expression of FoxA2 Using Synthetic mRNA Transfection of Rat NPCs (A) Generation of DA neurons by repeated mRNA transfection. Continuous Nurr1 mRNA (a and b), Nurr1 and eGFP mRNA (c and d), and Nurr1 and FoxA2 mRNA (e and f) transfection was performed into rat NPCs daily. These differentiated cells were fixed on differentiation days 3 and 7 (Diff. 3 and Diff. 7). Then, DA neurons were stained with anti-NURR1 and anti-TH antibodies. (B) TH⁺ cells were counted on differentiation days 3 and 7. Error bars represent SE from the results of three independent experiments. Paired t test was used to assess differences between two groups (***p < 0.001). The scale bar represents 20 μm.

Figure 4

Generation of Mature DA Neurons by Delayed Nurr1 and FoxA2 mRNA Transfection (A) Synthetic Nurr1 and FoxA2 mRNA were transfected daily into rat NPCs starting at differentiation day 7. mRNA-induced DA neurons were stained with anti-NURR1 and anti-TH on differentiation days 10, 16, 22, and 28. (B) TH⁺ cells were counted on differentiation days 10, 16, 22, and 28. (C) NURR1⁺ cells were counted on differentiation days 10, 16, 22, and 28. (D) The number of fibers in an individual TH⁺ cell was counted on differentiation days 10, 16, 22, and 28. (E) The lengths of TH⁺ cells’ fibers were measured on differentiation days 10, 16, 22, and 28. Error bars represent SE from the results of three independent experiments. Paired t test was used to assess differences between two groups (*p < 0.05, **p < 0.01, ***p < 0.001). The scale bar represents 20 μm.

Figure 5

Functional Analyses of Post-differentiated Nurr1 + FoxA2 mRNA-Induced DA Neurons (A) RT-PCR analysis of post-differentiated Nurr1 + FoxA2 mRNA-induced DA neurons (Nr[L]+Fr[L]). On differentiation day 15, Nr(L)+Fr(L) expressed DA neuron markers. (B) Quantitative real-time PCR analysis: Nr(L)+Fr(L). On differentiation day 15, Nr(L)+Fr(L) expressed functional DA neuron markers. (C) DA released from Nr(L)+Fr(L). On differentiation day 15, DA was released in early mRNA-induced DA neurons (Nr+Fr) and Nr(L)+Fr(L). Nr(L)+Fr(L) released higher DA compared with Nr+Fr. DA-released supernatants were harvested under two conditions: incubated for 24 hr or stimulated by 56 mM KCl for 30 min. (D) Electrophysiological analysis of Nr(L)+Fr(L). On differentiation day 22, the electrophysiological analysis showed active sodium current and action potential firing. Error bars represent SE from the results of three independent experiments. Paired t test was used to assess differences between two groups (*p < 0.05, **p < 0.01, ***p < 0.001).