Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs

Please cite this article in press as: Kim et al., Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs, Molecular Therapy (2017), http://dx.doi.org/10.1016/j.ymthe.2017.06.015

Original Article

Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs Sang-Mi Kim,1,4,9 Mi-Sun Lim,2,3,9 Eun-Hye Lee,1 Sung Jun Jung,4,5 Hee Yong Chung,4,6,7 Chun-Hyung Kim,8 and Chang-Hwan Park1,4,6,7 1Department

of Biomedical Science, Graduate School, Hanyang University, Seoul 04763, Korea; 2R&D Center, Jeil Pharmaceutical Co., Ltd., Yongin 17172, Korea; 3Institute

of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul 06360, Korea; 4Hanyang Biomedical Research Institute, Hanyang University, Seoul 04763, Korea; 5Department of Physiology, College of Medicine, Hanyang University, Seoul 04763, Korea; 6Department of Microbiology, College of Medicine, Hanyang University, Seoul 04763, Korea; 7Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Korea; 8Paean Biotechnology, Inc., Daejeon 34028, Korea

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.

INTRODUCTION Parkinson’s disease (PD) is a degenerative brain disorder of the CNS that is accompanied by akinesia, rigidity, and tremor. Currently, levodopa (L-dopa), a precursor of dopamine (DA), is the standard therapy for PD. However, treatment with L-dopa causes adverse effects such as hyperkinesia, dyskinesia, autonomic dysfunction, and sensory and pain symptoms. Because the main pathological feature of PD is the selective loss of DA neurons in the substantia nigra (SN), cell replacement therapy represents the logical next step for PD therapy. The proof of concept was demonstrated with fetal ventral mesencephalon (VM) cell transplantation, showing promising outcomes in both animal models and human patients.1 However, fetal VM cell transplantation for PD is fraught with ethical and technical hurdles. Therefore, the development of an ideal DA cell source for transplantation would be critical for the development of an effective cell therapy approach to PD.

Various methods to generate DA neurons were attempted such as directed differentiation from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to DA neurons,2–7 transdifferentiation by defined transcription factors from somatic cells to DA neurons,8–14 and overexpression of DA-fate inducing genes in neural precursor cells (NPCs).15–19 The resulting DA neurons improved behavioral deficits in PD animal models,20,21 indicating the potential for differentiated DA neurons as a cell source for PD cell therapy. Most DA neurons derived from somatic cells or NPCs were obtained by retroviral or lentiviral delivery of defined transcription factors or DA-fate inducing genes, leading to viral vector integration into the chromosome and genetic dysfunction. To meet the full potential of differentiated DA neurons, it is essential to establish an in vitro procedure to generate biomedically and clinically acceptable quality of differentiated DA neurons. Of the methods free of gene modification and mutagenesis, the use of synthetic mRNA transfection for an effective expression of genes of interest is well established.22–29 The mRNA-based gene delivery methods are integration free and result in rapid gene expression but suffer from some disadvantages, such as low efficiency of RNA transfection and instability of RNA. For this reason, mRNA transfection conditions were modified to increase mRNA transfection efficiency through various approaches, such as the development of

Received 31 January 2017; accepted 18 June 2017; http://dx.doi.org/10.1016/j.ymthe.2017.06.015. 9

These authors contributed equally to this work.

Correspondence: Hee Yong Chung, Graduate School of Biomedical Science and Engineering and Department of Microbiology, College of Medicine, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 04763, Korea. E-mail: [email protected] Correspondence: Chun-Hyung Kim, Paean Biotechnology, Inc., 160 Techno-2 St., Yuseong-gu, Daejeon 34028, Korea. E-mail: [email protected] Correspondence: Chang-Hwan Park, Laboratory of Neural Stem Cell Biology, Graduate School of Biomedical Science and Engineering and Department of Microbiology, College of Medicine, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 04763, Korea. E-mail: [email protected]

Molecular Therapy Vol. 25 No 9 September 2017 ª 2017 The American Society of Gene and Cell Therapy.

1

Please cite this article in press as: Kim et al., Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs, Molecular Therapy (2017), http://dx.doi.org/10.1016/j.ymthe.2017.06.015

Molecular Therapy

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 50 UTR, 30 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 mm.

mRNA-based method. We transcribed Nurr1 mRNA (N mRNA) and FoxA2 mRNA (F mRNA) in vitro, and the resulting mRNAs were purified and transfected into rat NPCs for NURR1 and FOXA2 protein expression. We found that the addition of cyclic AMP (cAMP) and its analog significantly improved protein expression efficiency. Finally, we confirmed that neurons obtained from N and F mRNA transfection of rat NPCs were functional and mature DA neurons by RT-PCR assay and real-time PCR assay of DA neuronal marker, DA release assay, and electrophysiological analysis.

DNA vector for RNA synthesis, the addition of capping, and poly A tailing for RNA stability and effective translation.30–34 Many key transcription factors and their functional roles during the DA neuron development process have been extensively and widely investigated.35,36 Nurr1 (NR4A2), a transcription factor belonging to the orphan nuclear receptor family, is an essential factor for midbrain-specific DA neuron development and can induce transplantable tyrosine hydroxylase (TH)+ DA neurons from rat NPCs.15–18,35,37–39 After transplantation of Nurr1-overexpressed rat NPCs into rat brains of PD rat model, DA neurons were found in the transplanted area.16,18 The additional expression of FoxA2 (winged helix/forkhead box A2: HNF3beta) further improved the yield, survival, and function of Nurr1-induced DA neurons.40–43 In this study, in order to generate clinically safe DA neurons from NPCs, we differentiated rat NPCs into DA neurons using an

2

Molecular Therapy Vol. 25 No 9 September 2017

The results of our study show that synthetic mRNA transfection is an efficient method of preparing functional DA neurons from NPCs, and the resulting DA neurons may serve as a novel cellular source for PD treatment.

RESULTS Protein Expression by Synthetic mRNA Transfection

Nurr1 and FoxA2 have essential roles in genesis, survival, and maintenance of DA neurons. On the basis of a previous report that co-expression of Nurr1 and FoxA2 in NPCs efficiently generated midbrain DA neurons,42 we decided to express NURR1and FOXA2 proteins by transfection of synthetic mRNA. This was accomplished by first creating a vector for mRNA synthesis, pcDNA/UTR120A, in which eGFP, FLAG-tagged Nurr1, or HA-tagged FoxA2 was inserted between the 50 and 30 UTRs (Figure 1A).22 Each mRNA, synthesized by in vitro transcription (IVT), was transfected into human embryonic kidney 293 (HEK293) cells and rat NPCs after purification (Figure S1). HEK293 cells were used as a positive control for comparison of mRNA transfection efficiency. Protein expressions were clearly

Please cite this article in press as: Kim et al., Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs, Molecular Therapy (2017), http://dx.doi.org/10.1016/j.ymthe.2017.06.015

www.moleculartherapy.org

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 mm.

observed in both HEK293 cells and rat NPCs after mRNA transfection. Immunocytochemical staining showed that 84% of HEK293 cells were eGFP+, 55% were NURR1+, and 28% were FOXA2+ (Figure 1B). However, protein expression in rat NPCs was significantly lower (26% eGFP+, 6% NURR1-positive, and 6% FOXA2+ population in rat NPCs; Figure 1B). To increase the expression efficiency in rat NPCs, we studied the effect of treatment with dibutyryl cAMP (dbcAMP), a cAMP derivative known to stimulate protein kinase A (PKA) signaling, leading to gene expression activation.44–46 When db-cAMP was added to mRNA-transfected rat NPCs, we observed the significant enhancement of protein expression in rat NPCs (Figures 1B–c, 1B–g, and 1B–k). In the presence of db-cAMP, the expression of eGFP, NURR1, and FOXA2 was increased by 1.8-, 2- and 2.3-fold, respectively. Taken together, these data show that proteins can be stably translated from in vitro synthesized mRNA and can be efficiently expressed in rat NPCs with the addition of db-cAMP. Nurr1 mRNA Induces TH Expression in Rat Embryonic Cortical NPCs

NURR1 protein has a short half-life, undergoing ubiquitin proteasome system (UPS)-mediated degradation in NPCs.47,48 In support of this, the NURR1 protein expression by synthetic mRNA transfec-

tion lasted at most 1 day because of rapid degradation in the rat NPCs, whereas eGFP expression was maintained for 3 days after mRNA transfection (Figure S2). To maintain the expression of NURR1 protein, we performed daily mRNA transfections (Figure 2).22 We performed immunocytochemistry analysis to detect the expression of TH, a marker specific to DA neurons, to determine whether Nurr1 overexpression effected rat NPCs differentiation into DA neurons (Figure 2A). As expected, TH+ cells appeared at differentiation day (Diff.) 1 and remained high at Diff. 3. However, the number of cells expressing TH began to decrease at Diff. 7 and almost disappeared by Diff. 10, probably because of cytotoxicity resulting from repetitive mRNA transfection (Figures 2B and 2C). FoxA2 and Nurr1 mRNA Synergistically Promote TH+ Cell Expression

FoxA2, a transcription factor expressed in the floor plate of the neural tube, plays an important role as co-activator of Nurr1-induced midbrain-specific DA neuron generation and can induce functional and survivable DA neurons.40,41,43,49,50 In order to test if FoxA2 has positive effects on DA neuron generation in rat NPCs, we co-expressed FOXA2 protein using FoxA2 retrovirus transduction and NURR1 protein using N mRNA transfection. Expectedly, FoxA2

Molecular Therapy Vol. 25 No 9 September 2017

3

Please cite this article in press as: Kim et al., Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs, Molecular Therapy (2017), http://dx.doi.org/10.1016/j.ymthe.2017.06.015

Molecular Therapy

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 mm.

increased N mRNA-induced NURR1 protein expression at both Diff. 3 and 7 (Figures S3A and S3B). In addition, the generation of TH+ cells was significantly increased by co-expression of FoxA2 and Nurr1 compared to the Nurr1 alone group (TH+ Diff. 3: empty virus + N mRNA [1.73 ± 0.31%], FoxA2 virus + N mRNA [15.89 ± 0.79%]), (TH+ Diff. 7: empty virus + N mRNA [1.73 ± 1.09%], FoxA2 virus + N mRNA [7.01 ± 2.13%]) (Figures S3A and S3C). On the basis of the results in Figure S3, we induced DA neurons with co-transfection of in vitro synthetic FoxA2 and N mRNAs. mRNA transfection of Nurr1 alone, Nurr1 and eGFP, and Nurr1 and FoxA2 into rat NPCs was performed daily, and differentiation of TH+ cells was scored at days 3 and 7. Again, we could observe that TH+ cells were increased by co-expression of Nurr1 and FoxA2 compared with expression of Nurr1 alone (N mRNA TH+: Diff. 3 [2.74 ± 0.21%], Diff. 7 [0.83 ± 0.09%]), (N mRNA + eGFP mRNA TH+: Diff. 3 [0.07 ± 0.01%], Diff. 7 [0.04 ± 0.03%]), (N mRNA + F mRNA TH+: Diff. 3 [6.82 ± 0.52%], Diff. 7 [5.107 ± 0.15%]) (Figure 3). Collectively, these results demonstrate that the efficient generation of DA neurons requires the co-expression of Nurr1 and FoxA2, and mRNA transfection is an efficient method of generating DA neurons from rat NPCs. Improved Survival of mRNA-Based DA Neurons Generated by mRNA Transfection through Delayed mRNA Transfection

On the basis of previous data showing that the delayed expression of Nurr1 efficiently induced mature and functional DA neurons,51

4

Molecular Therapy Vol. 25 No 9 September 2017

we attempted to delay the expression of Nurr1 and FoxA2 in order to generate mRNA-induced mature DA neurons. To do so, we began transfection of N and F mRNA at Diff. 7, and transfections were performed once per day thereafter. We fixed N and F mRNA-induced DA neurons on Diff. 10, 16, 22, and 28 and then performed immunocytochemistry analysis. Dissimilarly to early transfected cells, N + F mRNA delayed transfected DA neurons were maintained for up to 4 weeks after differentiation initiation (Figure 4A). In addition, the delayed N and F mRNA transfection resulted in the gradually increased number of DA neurons, and the differentiated DA neurons lasted longer (TH+: Diff. 10 [1.47 ± 0.21%], Diff. 16 [2.55 ± 0.04%], Diff. 22 [2.93 ± 0.34%], Diff. 28 [3.3 ± 0.26%]) (Figure 4B). Also, the increased number of DA neurons accompanied with the increased number of NURR1+ cells (NURR1+: Diff. 10 [11.93 ± 3.06%], Diff. 16 [17.7 ± 1.8%], Diff. 22 [16.88 ± 2.38%], Diff. 28 [19.4 ± 3%]) (Figure 4C). Moreover, the number and length of TH+ neuronal fibers were more consistently maintained in mature neurons during differentiation (TH+ fiber number: Diff. 10 [3.92 ± 0.39], Diff. 16 [5.29 ± 0.38], Diff. 22 [5.13 ± 0.46], Diff. 28 [4.89 ± 0.12]), (TH+ fiber length: Diff. 10 [169.28 ± 17.87 pixels], Diff. 16 [204.32 ± 4.91 pixels], Diff. 22 [212.78 ± 4.21 pixels], Diff. 28 [207.87 ± 6.23 pixels]) (Figures 4D and 4E). Thus, we clearly showed that delayed transfection of mRNAs is an efficient way of inducing more mature DA neurons.

Please cite this article in press as: Kim et al., Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs, Molecular Therapy (2017), http://dx.doi.org/10.1016/j.ymthe.2017.06.015

www.moleculartherapy.org

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 mm.

Acquisition of Functionality of N and F mRNA-Induced DA Neurons

In order to determine the functionality of the N and F mRNAinduced DA neurons, we performed RT-PCR, real-time PCR, DA release assay, and electrophysiological analysis (Figure 5). RT-PCR and real-time PCR data showed that N and F mRNA-induced DA neurons expressed DA-specific markers, including TH, amino acid aromatic decarboxylase (AADC), DA transporter (DAT), vesicular monoamine transporter 2 (VMAT2), and LIM homeobox transcrip-

tion factor 1, alpha (Lmx1A) (Figures 5A and 5B). Also, DA release assay clearly demonstrated that on Diff. 15, N and F mRNA-induced neuronal cells released high levels of DA during differentiation. In addition, DA release evoked by depolarization was observed in differentiated cells using 56 mM KCl stimulation (Figure 5B). Also, we performed electrophysiological analysis to assess the functionality of mRNA-induced DA neurons (Figure 5C). Through whole-cell patch clamp analysis, N and F mRNA-induced DA neurons produced a sodium current and showed action potential firing. These findings

Molecular Therapy Vol. 25 No 9 September 2017

5

Please cite this article in press as: Kim et al., Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs, Molecular Therapy (2017), http://dx.doi.org/10.1016/j.ymthe.2017.06.015

Molecular Therapy

collectively suggest that co-expression of NURR1 and FOXA2 proteins by transfection of in vitro synthesized mRNAs yielded functional DA neurons from rat NPCs.

DISCUSSION PD is a degenerative brain disorder that is caused mainly by gradual degeneration of DA neurons of the nigrostriatal pathway.52 To date, the various therapies used to treat PD provide only a transient mitigation of symptoms and have significant side effects. Previous experiments demonstrated a recovery in DA transport in patients who received transplants of DA neurons from human fetal brain tissue, which led to the study of cell replacement therapy for PD.53–55 On the basis of these studies, when rat NPCs overexpressing DA-fate inducing genes were transplanted into a rat PD model, the function and survival of the induced DA neurons were demonstrated in the transplanted areas.16,18,42 However, viral delivery of DA-fate inducing genes into NPCs may result in random integration into chromosomes. Therefore, we aimed to generate clinically safe DA neurons as cell therapy source using the mRNA-based method. To date, various reprogramming methods based on DNA, RNA, microRNA, and proteins have been used to obtain sources for nonintegrating cell replacement therapies. Among the reprogramming methods, mRNA transfection is the perfect non-integrating method because mRNAs have a short half-life and cannot integrate and replicate.56 Thus, the use of mRNA is a crucial step to generate clinicalgrade cell therapies. To this end, we tested protein expressions by immunocytochemistry after mRNA transfection to HEK293 cells and rat NPCs. Protein expression in HEK293 cells was significantly higher than that in rat NPCs, probably because of differences in transfection efficiency.57 In addition, protein expression by N and F mRNA, DA-fate inducing genes, was relatively low compared with protein expression by eGFP mRNA in both cells. This discrepancy in expression level may result from inherent gene properties such as different mRNA and protein turnover rate. Most housekeeping genes related to metabolism and structure have long mRNA half-lives, whereas genes with regulatory function have short half-lives.58 As transcription factors, N and F mRNAs have very short half-lives of less than 1 hr, and rapid mRNA decay may occur after transfection, resulting in low protein expression compared with eGFP, which has a long half-life. cAMP is a crucial regulator of gene expression, exerting its control transcriptionally and post-transcriptionally. The role of cAMP and its analog in the post-transcriptional pathway is to increase mRNA

Figure 5. Functional Analyses of Post-differentiated Nurr1 + FoxA2 mRNAInduced 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

6

Molecular Therapy Vol. 25 No 9 September 2017

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).

Please cite this article in press as: Kim et al., Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs, Molecular Therapy (2017), http://dx.doi.org/10.1016/j.ymthe.2017.06.015

www.moleculartherapy.org

half-life and stability.44–46 In this context, our expression data showed the prominent role of cAMP in protein expression by mRNA transfection method (Figures 1B–c, 1B–g, and 1B–k). Together, synthetic mRNA transfection in conjunction with supplementation with cAMP is a promising approach for DA-inducible gene expression. Studies looking at cell replacement therapy for PD established that Nurr1 is a crucial factor for induction of ventral midbrain-specific DA neurons. As dopaminergic fate regulator, Nurr1 is expressed in the ventral midbrain and induces TH expression at the post-mitotic stage of differentiation in ventral midbrain-specific DA neurons.36 Also, overexpression of Nurr1 in rodent NPCs induced transplantable DA neurons.15,42

schedule is an efficient and safe procedure to generate DA neurons from rat NPCs. We believe that the same protocol will be applicable with human NPCs and iPSCs, and we are currently testing the protocol. The advantages of mRNA-based methods include their simplicity, cell fate controllability, and the absence of the risk of gene modification and mutagenesis. Our results can serve as the foundation for future studies aiming to develop safe cell therapy for PD and other degenerative brain disorders. As the quest for the generation of safe DA neurons continues the efficacy and safety, DA by mRNA-based methods should be validated in transplantation studies.

MATERIALS AND METHODS Isolation of Rat NPCs

Based on the successful protein expression of dopaminergic fateinducing genes by mRNA transfection method (Figure 1), we next attempted to induce DA neurons from rat NPCs using Nurr1 alone. N mRNA transfection alone was insufficient to induce DA neurons, probably because of the short half-life of its mRNA and of the resulting protein. Next, we added F mRNA into rat NPCs with N mRNA. FoxA2 synergistically cooperates with Nurr1 to induce DA phenotype acquisition, midbrain-specific gene expression, and neuronal maturation.42 rat NPCs co-expressing Nurr1 and FoxA2 generated transplantable DA neurons with better yield, survival, and function.42,43 Also, we controlled transfection timing on the basis of a paper published in 2012 that suggested mature and functional DA neurons induced by delayed expression of Nurr1.51 On the basis of these two reports, we co-transfected N and F mRNAs into rat NPCs in the delayed schedule to investigate whether the modified transfection schedule would increase the efficiency of DA neuron generation in repeated mRNA transfection protocol. Our results showed that the delayed transfection of the N and F mRNA, compared with cDNA-expressing plasmid transfection or retroviral transduction, was superior in DA neuron generation. We also tested mRNA modification protocol, reported by Warren et al.22 That study demonstrated that repetitive transfection of synthetically modified mRNAs can efficiently reprogram human fibroblasts into human iPSCs.22 Modified mRNAs were synthesized by IVT with modified ribonucleotides such as 5-methylcytidine and pseudouridine. The use of modified mRNAs attenuated interferon signaling, as revealed by reduced expression of interferon response genes, interferon-a, interferon-b, interferoninduced protein with tetratricopeptide repeats 1, 20 -50 -oligoadenylate synthase 1, protein kinase R, retinoic acid inducible gene 1, and others, indicating reduced immunogenicity by bypassing innate anti-viral responses in the reprogramming process. As a result, conversion efficiencies and kinetics of human iPSCs using modified mRNAs were effectively increased than conversion efficiencies and kinetics of human iPSCs using viral vectors. However, when we used modified mRNA encoding DA-inducible genes, we could not clearly tell the difference between the normal and modified mRNA groups. Thus, we mainly used normal mRNAs to generate mRNAinduced DA neurons in this article. Taken together, we show, as a proof of concept, that repeated synthetic mRNA transfection of Nurr1 and FoxA2 in the delayed

Animals were housed and treated according to the Institutional Animal Care and Use Committee (IACUC, 2016-0194A) guidelines of Hanyang University. Rat NPCs were obtained from the cortex of a Sprague-Dawley (SD) rat embryo (embryonic day 14) (DaeHan BioLink). rat NPCs dissociated from the rat cortex tissues were cultured on a coated dish with 15 mg/mL poly-L-ornithine (PLO; Sigma-Aldrich) and 1 mg/mL fibronectin (FN; Sigma-Aldrich) at 37 C and 5% CO2. rat NPCs were then allowed to proliferate in N2 medium supplemented with 20 ng/mL basic fibroblast growth factor (bFGF; R&D Systems) and were differentiated in N2 medium supplemented with 0.2 mM ascorbic acid (Sigma-Aldrich), 20 ng/mL brainderived neurotrophic factor (BDNF; R&D Systems), 20 ng/mL glial cell line-derived neurotrophic factor (GDNF; R&D Systems), and 250 mg/mL dibutyryl-cAMP (db-cAMP; Sigma-Aldrich). Before transfection with the synthesized mRNA, cAMP derivatives, such as db-cAMP, 10 mM Forskolin (Sigma-Aldrich), and 5 mM NKH477 (Sigma-Aldrich), were treated into rat NPCs. After transfection with the synthesized mRNA, 200 ng/mL B18R (interferon-gamma inhibitor; eBioscience) was added to the rat NPCs. Plasmid Constructions

pcDNA/UTR120A, a vector for mRNA synthesis, was created by modification of plasmid pcDNA3.1(+) (Invitrogen). Some restriction enzyme sites (896–930 bp, 980–992 bp) of pcDNA3.1+ were replaced by restriction enzymes (NheI, BamHI, NotI, and XbaI). Synthesized 50 UTR, 50 UTR reverse, 30 UTR, 30 UTR reverse, 120 pA, and 120 pA reverse oligomers (IDT) were annealed and inserted behind the T7 promoter of pcDNA3.1(+). The oligonucleotide sequences are shown in Table S1.22 The genes of interest, such as eGFP, FLAG-tagged Nurr1, and HA-tagged FoxA2, were inserted between the 50 UTR and 30 UTR of the pcDNA/UTR120A. mRNA Synthesis

The eGFP/UTR120A, Nurr1(FLAG)/UTR120A, and FoxA2(HA)/ UTR120A constructs were linearized by a restriction enzyme, EcoRV (Takara Bio). The digested vectors, which were the template for mRNA synthesis, were synthesized into the mRNA by a MEGAscript T7 Kit (Ambion). The transcription mixture was incubated in vitro at 37 C for 2 hr. Modified mRNAs were generated by adding modified ribonucleotide, 50 -methylcytidine, and pseudouridine (Trilink Biotechnologies). Then, DNase was added at 37 C for 15 min. Next,

Molecular Therapy Vol. 25 No 9 September 2017

7

Please cite this article in press as: Kim et al., Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs, Molecular Therapy (2017), http://dx.doi.org/10.1016/j.ymthe.2017.06.015

Molecular Therapy

50 capping and poly A tailing were performed with the addition of the ScriptCap m7G Capping System, 20 -O-methyltransferase, and poly(A) polymerase (Epicenter, currently available from CELLSCRIPT) to the synthesized mRNA. Finally, the mRNAs were precipitated by 2.5 M ammonium acetate (Ambion) and dissolved in RNA Storage Solution (Ambion). The mRNAs were stored in a deep freezer at 70 C. mRNA Transfection

One day before transfection, NPCs (50,000 cells/Ø12 mm) were plated onto a slide glass in a PLO/FN pre-coated 24-well plate with expansion media without antibiotics. The synthesized mRNAs and the transfection reagent Lipofectamine 2000 (Invitrogen) were separately diluted in Opti-MEM media (Invitrogen) and then incubated at room temperature for 5 min. The mixtures were then combined and incubated at room temperature for 20 min. The incubated mixtures were used to treat the rat NPCs. After 3 hr, the mixtures were exchanged for the expansion media or the differentiation media added recombinant protein B18R. RT-PCR and Real-Time PCR

To synthesize cDNA, RNAs were extracted from the rat NPCs using TRI REAGENT (Molecular Research Center). Five micrograms of total RNA was used to generate cDNA using the Superscript Kit (Invitrogen). The cDNAs were used for amplification (sequences shown in Table S3), and the identity of the amplicons was confirmed by electrophoresis on 1.5% agarose gel. Real-time PCR analyses were conducted as described previously.9 Real-time PCR was conducted on a CFX96 real-time system using iQ SYBR Green Supermix (Bio-Rad). Realtime conditions were as follows: 60 C annealing temperature and 45 cycles. Real-time PCR primer sequences are indicated in Table S4. Immunocytochemistry

Cultured cells were fixed with 4% formaldehyde (Sigma-Aldrich). The fixed cells were blocked with 0.1% BSA/PBS, 10% normal goat serum (NGS; Pel-Freez), and 0.03% Triton X-100 (Sigma-Aldrich) for 1 hr. Primary antibodies were added to the blocked cells at 4 C overnight. The primary antibodies are shown in Table S2. After overnight incubation, the cells were labeled with biotin-conjugated secondary antibodies (Vector Laboratories) and fluorescence-labeled (DTAF or Rhodamin or Cy3) secondary antibodies (Jackson ImmunoResearch Laboratories). Cells were mounted onto glass slides by VECTASHIELD with DAPI (Vector Laboratories) mounting medium. The stained cells were visualized using an epifluorescence microscope (Leica Microsystems) or a confocal microscope (Leica Microsystems). The lengths of TH+ cells’ fibers were measured using the Leica Application Suite (LAS) image analysis package.

bromide; Sigma-Aldrich) was added to the virus supernatants and stored at 70 C. DA Release Assay

The DA release assay was performed using the Dopamine Research ELISA Kit (Labor Diagnostika Nord) according to the manufacturer’s instructions. On Diff. 15, the DA-released supernatants were collected under two conditions: incubated for 24 hr or stimulated by 56 mM KCl for 30 min. The DA level was calculated on the basis of the standard curve generated with the standard control. Electrophysiological Analysis

Whole-cell patch clamp recordings from rat NPCs were performed at room temperature (22 ± 1 C) using an EPC 10 USB amplifier (HEKA Elektronik). The resistances of the pipettes were 4–8 MU when filled with the solution, which was composed of 140 mM K-gluconate, 5 mM di-tris-phosphocreatine, 5 mM NaCl, 4 mM MgATP, 0.4 mM Na2GTP, 15 mM HEPES, and 2.5 mM Na-pyruvate, adjusted to pH 7.3 with KOH. Series resistance was compensated by 70%–80%, and currents were low-pass-filtered at 2 kHz and sampled at 10 kHz, with hold potential of 60 mV. The bath solution included 124 mM NaCl, 26 mM NaHCO3, 3.2 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 1.25 mM NaHPO4, and 10 mM glucose saturated with 95% O2 and 5% CO2. Cell Counting and Statistical Analysis

Cell counting was performed on randomly selected 10–15 microscopic fields per well and three wells per experimental conditions. Each experiment was independently performed at least three times. All data are expressed as mean ± SE. Using SigmaPlot for Windows version 10.0 (Systat Software), paired t tests were performed for statistical comparison of data.

SUPPLEMENTAL INFORMATION Supplemental Information includes three figures and four tables and can be found with this article online at http://dx.doi.org/10.1016/j. ymthe.2017.06.015.

AUTHOR CONTRIBUTIONS Conceptualization, S.-M.K., M.-S.L., C.-H.K., and C.-H.P.; Methodology, S.-M.K., E.-H.L., and S.J.J.; Investigation, S.-M.K., S.J.J., H.Y.C., C.-H.K., and C.-H.P.; Writing – Original Draft, S.-M.K. and M.-S.L.; Writing – Review & Editing, H.Y.C., C.-H.K., and C.-H.P.; Funding Acquisition, C.-H.K. and C.-H.P.; Resources, C.-H.P.; Supervision, H.Y.C., C.-H.K., and C.-H.P.

Production of Recombinant Retrovirus

ACKNOWLEDGMENTS

We used the retroviral vector pCL, as described in a previous study.17 A retroviral construct encoding HA-tagged FoxA2 or an empty retroviral construct was transfected into 293GPG packaging cells using the transfection reagent Lipofectamine 2000. After 72 hr, the virus supernatant was collected for 10 days. Polybrene, 2 mg/mL (hexadimethrine

We thank Dr. Steven F. Dowdy of the University of California, San Diego, School of Medicine, for his assistance with the synthesis and isolation of RNA. We also thank Dr. Pierre Leblanc of the McLean Hospital, Molecular Neurobiology Laboratory, for his very careful review of our manuscript. This work was supported by the Bio & Medical

8

Molecular Therapy Vol. 25 No 9 September 2017

Please cite this article in press as: Kim et al., Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs, Molecular Therapy (2017), http://dx.doi.org/10.1016/j.ymthe.2017.06.015

www.moleculartherapy.org

Technology Development Program (NRF-2016M3A9B4918833) and the Basic Science Research Program (NRF-2016R1A2B4007640) through the National Research Foundation of Korea and the Korea Health Technology R&D Projectthrough the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (grant HI16C1013).

REFERENCES 1. Lindvall, O., and Björklund, A. (2004). Cell therapy in Parkinson’s disease. NeuroRx 1, 382–393. 2. Park, C.H., Minn, Y.K., Lee, J.Y., Choi, D.H., Chang, M.Y., Shim, J.W., Ko, J.Y., Koh, H.C., Kang, M.J., Kang, J.S., et al. (2005). In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons. J. Neurochem. 92, 1265–1276. 3. Ko, J.Y., Park, C.H., Koh, H.C., Cho, Y.H., Kyhm, J.H., Kim, Y.S., Lee, I., Lee, Y.S., and Lee, S.H. (2007). Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons. J. Neurochem. 103, 1417–1429. 4. Kriks, S., Shim, J.W., Piao, J., Ganat, Y.M., Wakeman, D.R., Xie, Z., Carrillo-Reid, L., Auyeung, G., Antonacci, C., Buch, A., et al. (2011). Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 480, 547–551. 5. Cho, M.S., Lee, Y.E., Kim, J.Y., Chung, S., Cho, Y.H., Kim, D.S., Kang, S.M., Lee, H., Kim, M.H., Kim, J.H., et al. (2008). Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. U S A 105, 3392–3397. 6. Rhee, Y.H., Ko, J.Y., Chang, M.Y., Yi, S.H., Kim, D., Kim, C.H., Shim, J.W., Jo, A.Y., Kim, B.W., Lee, H., et al. (2011). Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease. J. Clin. Invest. 121, 2326–2335. 7. Sundberg, M., Bogetofte, H., Lawson, T., Jansson, J., Smith, G., Astradsson, A., Moore, M., Osborn, T., Cooper, O., Spealman, R., et al. (2013). Improved cell therapy protocols for Parkinson’s disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons. Stem Cells 31, 1548–1562. 8. Kim, J., Su, S.C., Wang, H., Cheng, A.W., Cassady, J.P., Lodato, M.A., Lengner, C.J., Chung, C.Y., Dawlaty, M.M., Tsai, L.H., and Jaenisch, R. (2011). Functional integration of dopaminergic neurons directly converted from mouse fibroblasts. Cell Stem Cell 9, 413–419. 9. Lim, M.S., Chang, M.Y., Kim, S.M., Yi, S.H., Suh-Kim, H., Jung, S.J., Kim, M.J., Kim, J.H., Lee, Y.S., Lee, S.Y., et al. (2015). Generation of dopamine neurons from rodent fibroblasts through the expandable neural precursor cell stage. J. Biol. Chem. 290, 17401–17414. 10. Lim, M.S., Lee, S.Y., and Park, C.H. (2015). FGF8 is essential for functionality of induced neural precursor cell-derived dopaminergic neurons. Int. J. Stem Cells 8, 228–234. 11. Sheng, C., Zheng, Q., Wu, J., Xu, Z., Sang, L., Wang, L., Guo, C., Zhu, W., Tong, M., Liu, L., et al. (2012). Generation of dopaminergic neurons directly from mouse fibroblasts and fibroblast-derived neural progenitors. Cell Res. 22, 769–772.

16. Park, C.H., Kang, J.S., Shin, Y.H., Chang, M.Y., Chung, S., Koh, H.C., Zhu, M.H., Oh, S.B., Lee, Y.S., Panagiotakos, G., et al. (2006). Acquisition of in vitro and in vivo functionality of Nurr1-induced dopamine neurons. FASEB J. 20, 2553–2555. 17. Park, C.H., Kang, J.S., Yoon, E.H., Shim, J.W., Suh-Kim, H., and Lee, S.H. (2008). Proneural bHLH neurogenin 2 differentially regulates Nurr1-induced dopamine neuron differentiation in rat and mouse neural precursor cells in vitro. FEBS Lett. 582, 537–542. 18. Shim, J.W., Park, C.H., Bae, Y.C., Bae, J.Y., Chung, S., Chang, M.Y., Koh, H.C., Lee, H.S., Hwang, S.J., Lee, K.H., et al. (2007). Generation of functional dopamine neurons from neural precursor cells isolated from the subventricular zone and white matter of the adult rat brain using Nurr1 overexpression. Stem Cells 25, 1252–1262. 19. Park, C.H., Kang, J.S., Kim, J.S., Chung, S., Koh, J.Y., Yoon, E.H., Jo, A.Y., Chang, M.Y., Koh, H.C., Hwang, S., et al. (2006). Differential actions of the proneural genes encoding Mash1 and neurogenins in Nurr1-induced dopamine neuron differentiation. J. Cell Sci. 119, 2310–2320. 20. Bjorklund, L.M., Sánchez-Pernaute, R., Chung, S., Andersson, T., Chen, I.Y., McNaught, K.S., Brownell, A.L., Jenkins, B.G., Wahlestedt, C., Kim, K.S., and Isacson, O. (2002). Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc. Natl. Acad. Sci. U S A 99, 2344–2349. 21. Kim, J.H., Auerbach, J.M., Rodríguez-Gómez, J.A., Velasco, I., Gavin, D., Lumelsky, N., Lee, S.H., Nguyen, J., Sánchez-Pernaute, R., Bankiewicz, K., and McKay, R. (2002). Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 418, 50–56. 22. Warren, L., Manos, P.D., Ahfeldt, T., Loh, Y.H., Li, H., Lau, F., Ebina, W., Mandal, P.K., Smith, Z.D., Meissner, A., et al. (2010). Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618–630. 23. Plews, J.R., Li, J., Jones, M., Moore, H.D., Mason, C., Andrews, P.W., and Na, J. (2010). Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach. PLoS ONE 5, e14397. 24. Tavernier, G., Wolfrum, K., Demeester, J., De Smedt, S.C., Adjaye, J., and Rejman, J. (2012). Activation of pluripotency-associated genes in mouse embryonic fibroblasts by non-viral transfection with in vitro-derived mRNAs encoding Oct4, Sox2, Klf4 and cMyc. Biomaterials 33, 412–417. 25. Angel, M., and Yanik, M.F. (2010). Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins. PLoS ONE 5, e11756. 26. Yoshioka, N., Gros, E., Li, H.R., Kumar, S., Deacon, D.C., Maron, C., Muotri, A.R., Chi, N.C., Fu, X.D., Yu, B.D., and Dowdy, S.F. (2013). Efficient generation of human iPSCs by a synthetic self-replicative RNA. Cell Stem Cell 13, 246–254. 27. Simeonov, K.P., and Uppal, H. (2014). Direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mRNAs. PLoS ONE 9, e100134. 28. Hansson, M.L., Albert, S., González Somermeyer, L., Peco, R., Mejía-Ramírez, E., Montserrat, N., and Izpisua Belmonte, J.C. (2015). Efficient delivery and functional expression of transfected modified mRNA in human embryonic stem cell-derived retinal pigmented epithelial cells. J. Biol. Chem. 290, 5661–5672. 29. McLenachan, S., Zhang, D., Palomo, A.B., Edel, M.J., and Chen, F.K. (2013). mRNA transfection of mouse and human neural stem cell cultures. PLoS ONE 8, e83596.

12. Caiazzo, M., Dell’Anno, M.T., Dvoretskova, E., Lazarevic, D., Taverna, S., Leo, D., Sotnikova, T.D., Menegon, A., Roncaglia, P., Colciago, G., et al. (2011). Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476, 224–227.

30. Elango, N., Elango, S., Shivshankar, P., and Katz, M.S. (2005). Optimized transfection of mRNA transcribed from a d(A/T)100 tail-containing vector. Biochem. Biophys. Res. Commun. 330, 958–966.

13. Pfisterer, U., Kirkeby, A., Torper, O., Wood, J., Nelander, J., Dufour, A., Björklund, A., Lindvall, O., Jakobsson, J., and Parmar, M. (2011). Direct conversion of human fibroblasts to dopaminergic neurons. Proc. Natl. Acad. Sci. U S A 108, 10343–10348.

31. Hornung, V., Ellegast, J., Kim, S., Brzózka, K., Jung, A., Kato, H., Poeck, H., Akira, S., Conzelmann, K.K., Schlee, M., et al. (2006). 50 -Triphosphate RNA is the ligand for RIG-I. Science 314, 994–997.

14. Liu, X., Li, F., Stubblefield, E.A., Blanchard, B., Richards, T.L., Larson, G.A., He, Y., Huang, Q., Tan, A.C., Zhang, D., et al. (2012). Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells. Cell Res. 22, 321–332.

32. Holtkamp, S., Kreiter, S., Selmi, A., Simon, P., Koslowski, M., Huber, C., Türeci, O., and Sahin, U. (2006). Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108, 4009–4017.

15. Kim, J.Y., Koh, H.C., Lee, J.Y., Chang, M.Y., Kim, Y.C., Chung, H.Y., Son, H., Lee, Y.S., Studer, L., McKay, R., and Lee, S.H. (2003). Dopaminergic neuronal differentiation from rat embryonic neural precursors by Nurr1 overexpression. J. Neurochem. 85, 1443–1454.

33. Karikó, K., and Weissman, D. (2007). Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development. Curr. Opin. Drug Discov. Devel. 10, 523–532.

Molecular Therapy Vol. 25 No 9 September 2017

9

Please cite this article in press as: Kim et al., Efficient Generation of Dopamine Neurons by Synthetic Transcription Factor mRNAs, Molecular Therapy (2017), http://dx.doi.org/10.1016/j.ymthe.2017.06.015

Molecular Therapy

34. Van Tendeloo, V.F., Ponsaerts, P., Lardon, F., Nijs, G., Lenjou, M., Van Broeckhoven, C., Van Bockstaele, D.R., and Berneman, Z.N. (2001). Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood 98, 49–56. 35. Ang, S.L. (2006). Transcriptional control of midbrain dopaminergic neuron development. Development 133, 3499–3506. 36. Gale, E., and Li, M. (2008). Midbrain dopaminergic neuron fate specification: Of mice and embryonic stem cells. Mol. Brain 1, 8. 37. Thérond, P.P., Limbourg Bouchon, B., Gallet, A., Dussilol, F., Pietri, T., van den Heuvel, M., and Tricoire, H. (1999). Differential requirements of the fused kinase for hedgehog signalling in the Drosophila embryo. Development 126, 4039–4051. 38. Prakash, N., and Wurst, W. (2006). Genetic networks controlling the development of midbrain dopaminergic neurons. J. Physiol. 575, 403–410. 39. Perlmann, T., and Wallén-Mackenzie, A. (2004). Nurr1, an orphan nuclear receptor with essential functions in developing dopamine cells. Cell Tissue Res. 318, 45–52. 40. Ferri, A.L., Lin, W., Mavromatakis, Y.E., Wang, J.C., Sasaki, H., Whitsett, J.A., and Ang, S.L. (2007). Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner. Development 134, 2761–2769. 41. Kittappa, R., Chang, W.W., Awatramani, R.B., and McKay, R.D. (2007). The foxa2 gene controls the birth and spontaneous degeneration of dopamine neurons in old age. PLoS Biol. 5, e325. 42. Lee, H.S., Bae, E.J., Yi, S.H., Shim, J.W., Jo, A.Y., Kang, J.S., Yoon, E.H., Rhee, Y.H., Park, C.H., Koh, H.C., et al. (2010). Foxa2 and Nurr1 synergistically yield A9 nigral dopamine neurons exhibiting improved differentiation, function, and cell survival. Stem Cells 28, 501–512. 43. Yi, S.H., He, X.B., Rhee, Y.H., Park, C.H., Takizawa, T., Nakashima, K., and Lee, S.H. (2014). Foxa2 acts as a co-activator potentiating expression of the Nurr1-induced DA phenotype via epigenetic regulation. Development 141, 761–772. 44. Morris, B.J., Adams, D.J., Beveridge, D.J., van der Weyden, L., Mangs, H., and Leedman, P.J. (2004). cAMP controls human renin mRNA stability via specific RNA-binding proteins. Acta Physiol. Scand. 181, 369–373.

47. Alvarez-Castelao, B., Losada, F., Ahicart, P., and Castaño, J.G. (2013). The N-terminal region of Nurr1 (a.a 1-31) is essential for its efficient degradation by the ubiquitin proteasome pathway. PLoS ONE 8, e55999. 48. Jo, A.Y., Kim, M.Y., Lee, H.S., Rhee, Y.H., Lee, J.E., Baek, K.H., Park, C.H., Koh, H.C., Shin, I., Lee, Y.S., and Lee, S.H. (2009). Generation of dopamine neurons with improved cell survival and phenotype maintenance using a degradation-resistant nurr1 mutant. Stem Cells 27, 2238–2246. 49. Pospischil, A., Djamei, V., Rütten, M., Sydler, T., and Vaughan, L. (2007). Introduction to the Swiss way of teaching veterinary pathology in the twenty-first century: application of e-learning modules. J. Vet. Med. Educ. 34, 445–449. 50. Aufderheide, T.P., Alexander, C., Lick, C., Myers, B., Romig, L., Vartanian, L., Stothert, J., McKnite, S., Matsuura, T., Yannopoulos, D., and Lurie, K. (2008). From laboratory science to six emergency medical services systems: New understanding of the physiology of cardiopulmonary resuscitation increases survival rates after cardiac arrest. Crit. Care Med. 36 (11, Suppl), S397–S404. 51. Park, C.H., Lim, M.S., Rhee, Y.H., Yi, S.H., Kim, B.K., Shim, J.W., Kim, Y.H., Jung, S.J., and Lee, S.H. (2012). In vitro generation of mature dopamine neurons by decreasing and delaying the expression of exogenous Nurr1. Development 139, 2447–2451. 52. Lindvall, O., and Kokaia, Z. (2009). Prospects of stem cell therapy for replacing dopamine neurons in Parkinson’s disease. Trends Pharmacol. Sci. 30, 260–267. 53. Piccini, P., Brooks, D.J., Björklund, A., Gunn, R.N., Grasby, P.M., Rimoldi, O., Brundin, P., Hagell, P., Rehncrona, S., Widner, H., and Lindvall, O. (1999). Dopamine release from nigral transplants visualized in vivo in a Parkinson’s patient. Nat. Neurosci. 2, 1137–1140. 54. Wenning, G.K., Odin, P., Morrish, P., Rehncrona, S., Widner, H., Brundin, P., Rothwell, J.C., Brown, R., Gustavii, B., Hagell, P., et al. (1997). Short- and longterm survival and function of unilateral intrastriatal dopaminergic grafts in Parkinson’s disease. Ann. Neurol. 42, 95–107. 55. Lindvall, O. (1998). Update on fetal transplantation: the Swedish experience. Mov. Disord. 13 (Suppl 1 ), 83–87. 56. Schlaeger, T.M., Daheron, L., Brickler, T.R., Entwisle, S., Chan, K., Cianci, A., DeVine, A., Ettenger, A., Fitzgerald, K., Godfrey, M., et al. (2015). A comparison of non-integrating reprogramming methods. Nat. Biotechnol. 33, 58–63.

45. Dong, Y., Aronsson, M., Gustafsson, J.A., and Okret, S. (1989). The mechanism of cAMP-induced glucocorticoid receptor expression. Correlation to cellular glucocorticoid response. J. Biol. Chem. 264, 13679–13683.

57. Kim, Y.C., Shim, J.W., Oh, Y.J., Son, H., Lee, Y.S., and Lee, S.H. (2002). Co-transfection with cDNA encoding the Bcl family of anti-apoptotic proteins improves the efficiency of transfection in primary fetal neural stem cells. J. Neurosci. Methods 117, 153–158.

46. Cornelius, P., Marlowe, M., Call, K., and Pekala, P.H. (1991). Regulation of glucose transport as well as glucose transporter and immediate early gene expression in 3T3-L1 preadipocytes by 8-bromo-cAMP. J. Cell. Physiol. 146, 298–308.

58. Sharova, L.V., Sharov, A.A., Nedorezov, T., Piao, Y., Shaik, N., and Ko, M.S. (2009). Database for mRNA half-life of 19 977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells. DNA Res. 16, 45–58.

10

Molecular Therapy Vol. 25 No 9 September 2017