Functional characterization of rhesus embryonic stem cell-derived serotonin neurons

Abstract

Optimal function of the serotonin system is essential for mental health and its role in psychopathologies is undisputed. Enhancing the ability to study primate serotonin neurons in culture would facilitate understanding of intracellular signaling pathways that mediate the action of drugs and other epigenetic or developmental factors impacting human mental health. We were the first group to report differentiation of the non-human primate rhesus monkey embryonic stem cell (ESC) line 366.4 into cultures of serotonin neurons. In this study, we optimized yield and obtained functional characteristics of the derived serotonin neurons. Sequential treatments of ESC 366.4 during expansion stage with fibroblast growth factor 4 and sonic hedgehog markedly increased the yield of serotonin neurons. These serotonin neurons propagated action potentials and expressed GABA receptors. Also, for the first time we demonstrate that these ESC-derived serotonin neurons exhibit functional high-affinity transporter sites, as well as high-affinity 5HT_1A binding sites, which are essential targets of common psychoactive drugs. Finally, to test the generality of this method, we utilized another rhesus ESC line, ORMES-22, which efficiently differentiated into serotonin neurons. Together, these findings demonstrate the feasibility of our protocol to direct different primate ESC lines to serotonin neurons with physiological characteristics, which makes them a useful in vitro model system.

Keywords

embryonic stem cells differentiation serotonin rhesus macaque serotonin reuptake transporter

Introduction

The serotonin neural system plays a pivotal role in mood, stress sensitivity, integrative cognition and other autonomic functions. Serotonin functions through many different serotonin receptors, and it is important during development for guidance of neurogenesis, ultimate structure of the central nervous system (CNS) and neuronal plasticity.^1,2 Dysfunction of the serotonin system has been implicated in many psychopathologies such as depression, anxiety, schizophrenia, bipolar disorder, obsessive–compulsive disorder and eating disorders. Various drugs such as serotonin reuptake inhibitors, monoamine oxidase inhibitors and receptor antagonists have been designed for treatment; however, these medications only partially alleviate the symptoms and have numerous undesirable side-effects.^3,4 Intracellular signaling cascades, neurotransmitter synthesis, trafficking and release in primate serotonin neurons, as well as the effects of drugs on these events, have been unavailable for detailed study in vitro. The availability of a higher primate serotonin cell line would greatly facilitate study of the serotonin neural system at the molecular level in a way that would be relevant to human mental health.

Embryonic stem cells (ESCs) are unique pluripotent cells that can proliferate indefinitely in an undifferentiated state and are theoretically capable of differentiating into any cell type.⁵ Our laboratory was the first group to report the utilization of non-human primate rhesus monkey ESC 366.4 to differentiate into predominantly serotonin neurons⁶ in the hope of providing a cell culture model for cellular and molecular studies, as well as generating neurons for experimental transplantation into monkeys. The ability to experimentally put rhesus stem cells into the brain of rhesus monkeys provides a unique preclinical approach that cannot be currently attempted with humans.

Our ESC-derived serotonin cultures were previously shown to express serotonergic markers such as tryptophan hydroxylase (TPH), serotonin, serotonin reuptake transporters (SERT) and 5HT_1A autoreceptors, in addition to synthesizing serotonin, which was measured by enzyme-linked immunosorbent assays.⁶ However, the attractive pluripotent characteristics of stem cells also present significant challenges since they do not necessarily generate a single type of neuron (i.e. a pure culture). Other studies using mouse ESCs were able to derive cultures containing 25–60% serotonergic neurons.^7,8 Recently, Kumar et al. ⁹ showed differentiation of serotonin neurons from human ESCs with ∼50% differentiation rate, highlighting the difficulty of obtaining a pure culture. The goal of this study was to improve our previous protocol in order to optimize yield and to obtain physiological characterization of the ESC-derived serotonin neurons. Different combinations of mitogenic signaling molecules, specifically fibroblast growth factor 4 (FGF4) with sonic hedgehog (SHH), improved the yield of the serotonin neurons. These ESC-derived serotonin neurons have neuronal membrane properties and express specific functional SERT and 5HT_1A autoreceptors, thus making them a useful in vitro model. The feasibility of our new optimized protocol was shown by using another rhesus ESC line ORMES-22, which efficiently differentiated to serotonin neurons.

Materials and methods

ESC culture and in vitro differentiation

The rhesus monkey ESC line 366.4 was obtained from Dr James Thomson (Wisconsin National Primate Research Center). This line was derived from an in vivo flushed preimplantation embryo and has been characterized for its pluripotency, including its potential to differentiate into cells of the neural lineage.¹⁰ The rhesus monkey ESC line ORMES-22 was derived by Dr Shoukhrat Mitalipov (ART Core, Oregon National Primate Research Center).¹¹

ESCs were propagated and maintained as previously described.⁶ Briefly, ESCs were co-cultured with mitotically inactive (mitomycin C-treated; 1 mg/mL at 37°C for 30 min; Sigma-Aldrich, St Louis, MO, USA) mouse embryonic fibroblasts (MEFs). ESC culture medium consisted of 85% Dulbecco's modified Eagle's medium (DMEM/F12) supplemented with 1% non-essential amino acids, 2 mmol/L glutamine, 0.1 mmol/L β-mercaptoethanol (Sigma-Aldrich) and 15% fetal bovine serum (Thermo Fisher Scientific Inc, Waltham, MA, USA). ESCs and their colonies were observed daily and passaged every 6–8 d when colonies reached 1–1.5 mm in diameter. The pluripotency of ESCs was evaluated periodically by immunocytochemistry (ICC) with antibodies to Oct-4, stage-specific embryonic antigens (SSEA-3 and 4) and embryonic proteoglycans (TRA-1–60 and TRA-1–81) as described previously.⁶

A protocol consisting of multiple sequential steps was used to induce differentiation as follows:

Isolation of ESC colonies and formation of embryoid bodies (EBs): After ESC colonies attained a 1–1.5 mm diameter, they were isolated mechanically, triturated into intermediate-sized clumps (>200 cells/clump), transferred into 60 mm dishes (BD Biosciences, San Jose, CA, USA) and cultured in ESC medium at 37°C for 7 d. EBs were defined as Oct-4 negative, three-dimensional structures that could potentially give rise to endo-, ecto- and mesodermal cell lineages;

Selection (N1 stage): After formation of EBs, ESC medium was replaced with a selection medium composed of DMEM/Nutrient Mixture F12 (1:1) containing l-glutamine, sodium bicarbonate, pyridoxine hydrochloride, 1% ITSX (1 g/L insulin; 0.67 mg/L sodium selenite; 0.55 g/L transferrin and 0.2 g/L ethanolamine) and human plasma fibronectin (5 μg/mL). EBs were cultured in selection medium for 7 d and at this stage referred as neurospheres;

Expansion (N2 stage): At the end of the selection period, neurospheres were cultured in expansion medium composed of DMEM/Nutrient Mixture F12 (1:1), 1% N2 supplement (500 μg/mL insulin; 10,000 μg/mL transferrin; 0.63 μg/mL progesterone; 1611 μg/mL putrescine and 0.52 μg/mL selenite) and FGF4 (10 ng/mL) for 2 d supplemented with SHH (50 ng/mL) for an additional 5 d changing the medium daily;

Maturation of differentiated neural cells (N3 stage): Expanded neurospheres were gently dispersed into single-cell suspension using TrypLE (Invitrogen, Carlsbad, CA, USA) and then plated at 90% confluency on growth factor reduced (GFR)-matrigel-coated coverslips or wells depending on the application. Cells were cultured in Neurobasal medium with N2 and B27 supplement (Invitrogen) up to two months.

Immunocytochemistry

Cells grown on coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were permeabilized with 0.2% Triton-X and 0.1% Tween-20 in phosphate-buffered saline (PBS) for 40 min, followed by incubation with blocking solution (10% normal goat serum in PBS) for one hour. Cells were then incubated overnight at 4°C with primary rabbit polyclonal anti-TPH2 antibody (catalog#: NB100-74555; immunizing peptide corresponds to residues 15–30 of human TPH2, Novus Biologicals Inc, Littleton, CO, USA) and antibody–antigen complexes were detected with Alexa Fluor 488 secondary antibody (1:1000, Invitrogen) by incubation for one hour at room temperature. The samples were washed five times with PBS after each incubation, and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI).

Treatment with different mitogenic factors and SHH

To improve the viability and yield of serotonin neurons, the ESCs were treated with N2 medium (see above) during the expansion stage (N2 stage) with the following:

N2 media with 10 ng/mL FGF2 (Sigma-Aldrich) for 7 d;

N2 media with 10 ng/mL FGF2 for 2 d then addition of 50 ng/mL SHH (R&D Systems, Minneapolis, MN, USA) for 5 d;

N2 media with 10 ng/mL FGF4 (Sigma-Aldrich) for 7 d;

N2 media with 10 ng/mL FGF4 for 2 d then addition of 50 ng/mL SHH for 5 d.

The cells were immunostained for TPH2 with a rabbit antibody generated against human TPH2 followed by development with an anti-rabbit mouse monoclonal antibody conjugated to Alexa Fluor-488. The cultures were photographed at a low magnification (10×) using the Marianas stereological workstation (Intelligent Imaging Innovations, Denver, CO, USA). The Marianas stereological workstation with Slidebook 4.2 was used to obtain a montage and for analysis. The green-labeled, TPH2-positive pixel area and the DAPI-positive pixel area, indicating the total number of cells, were both reported in square microns (μ ² positive area). Two montages from two independent experiments for each treatment group (total of four montages) were analyzed. The TPH2-positive pixel area was divided by the DAPI-positive pixel area in each montage to obtain a ratio of TPH2 labeled neurons to total cells in the differentiated culture (n = 4/treatment). Differences between the treatment groups were determined by ANOVA followed by Newman–Keuls post-test using Prism Statistical software (Graph-Pad Software, Inc, San Diego, CA, USA). P < 0.05 was considered statistically significant.

Electrophysiology

Whole-cell patch-clamp recordings were made with electrodes pulled to 2–4 MΩ resistance filled with an internal solution (138 mmol/L potassium methylsulfate, 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) [HEPES]; 10 mmol/L KCl, 1 mmol/L MgCl₂, 1 mmol/L ethylene gylcol tetraacetic acid, 0.3 mmol/L CaCl₂, 4 mmol/L MgATP, 3 mmol/L NaGTP, pH 7.4). Cells were superfused with an external solution (146 mmol/L NaCl, 30 mmol/L dextrose, 5 mmol/L KCl, 5 mmol/L HEPES, 2.5 mmol/L CaCl₂, 1.2 mmol/L MgCl₂, pH 7.35). Junction potentials were calculated (JPCalc, Molecular Devices, Sunnyvale, CA, USA) and corrected at the beginning of the experiments. Capacitance and series resistance compensation (>80%) were corrected and data were collected with a Multiclamp amplifier (Molecular Devices) at 10 kHz and filtered with a low-pass Bessel filter at 2 kHz. Currents were digitized with Digidata1322 (Molecular Devices), collected and analyzed using Axograph (Axograph Scientific, Sydney, Australia). Action currents were elicited with a depolarizing step from −70 to −30 mV. Current–voltage plots were generated from a series of 10 mV steps from −100 to +20 mV. Drugs were diluted in appropriate buffers in concentrated stock solutions, then diluted further in external solution to final concentration. Drugs were applied by bath superfusion.

SERT-binding assay

To determine whether the ESC-derived serotonin neurons manifested a specific serotonin reuptake-binding site (SERT), a binding assay was conducted. The SERT-binding assay was adapted from Lu et al. ¹² Briefly, N3 stage serotonin neurons were preincubated in binding assay buffer (50 mmol/L Tris-HCl, 120 mmol/L NaCl, 5 mmol/L KCl, pH 7.4) for 15 min at room temperature. A volume of 2 μmol/L [³H]-paroxetine (15 Ci/mmol, Perkin-Elmer Life Sciences, Waltham, MA, USA) alone or in combination with 1 μmol/L Fluoxetine (Eli Lilly, Indianapolis, IN, USA) was added for 1.5 h at room temperature. To further verify the specificity, 1 μmol/L mazindol (Sigma-Aldrich), a noradrenaline reuptake inhibitor, was also added along with [³H]-paroxetine. Cells were collected in 100 μL of binding assay buffer and radioactivity was measured using a liquid scintillation counter (LS6000IC, Beckman, Brea, CA, USA). Differences between the treatment groups were determined by ANOVA followed by Newman–Keuls post-test using Prism Statistical software (Graph-Pad Software Inc) (n = 6/treatment). P < 0.05 was considered statistically significant.

5HT_1A-binding assay

To determine whether the ESC-derived serotonin neurons manifested a specific 5HT_1A autoreceptor, a binding assay was conducted. The 5HT_1A autoreceptor-binding assay was adapted from Lu and Bethea.¹³ Briefly, N3 stage serotonin neurons were incubated in preincubation buffer (170 mmol/L Tris-HCl, 4 mmol/L CaCl₂, pH 7.6) for 15 min at room temperature. Then, the cultures were incubated in binding assay buffer (preincubation buffer plus 0.01% l-ascorbic acid, 10 μmol/L pargyline [Sigma-Aldrich], 1 μmol/L fluoxetine containing 2 nmol/L [³H]-8-OH-DPAT [124.9 Ci/mmol, Perkin-Elmer Life Sciences] alone, or in combination with either 2 μmol/L cold 8-OH-DPAT or 2 μmol/L serotonin (Sigma-Aldrich) for 1.5 h at room temperature. Cells were collected in 100 µL of incubation binding assay buffer and radioactivity was measured using a liquid scintillation counter (LS6000IC, Beckman). Differences between the treatment groups were determined by ANOVA followed by Newman–Keuls post-test using Prism Statistical software (Graph-Pad Software, Inc) (n = 6/treatment). P < 0.05 was considered statistically significant.

Results

Improvement in viability and yield of serotonin neurons

Previously, we found that exposure of neurospheres to FGF2 helped differentiate the rhesus 366.4 ESC into serotonin neurons. These cultures were extensively examined by ICC and found to express markers that typify serotonergic neurons of the dorsal raphe of the monkey brain including TPH, serotonin and SERT.⁶ In order to improve the yield of monkey serotonin neurons, we cultured neurospheres in expansion (N2) medium with different combinations of FGF2 or FGF4 plus or minus SHH after three days for a total of seven days (Figure 1A). Figure 1C shows low-power montages of cultures that were immunolabeled for tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme for serotonin synthesis,¹⁴ and illustrate the colonies of TPH2-positive serotonin neurons. In higher magnification, these TPH2-positive neurons showed a typical serotonergic morphology as previously observed with extensive neurite outgrowth.^6,8,15 TPH2-positive pixel area was reported in square microns (μ ² positive area) along with DAPI-positive pixel area in square microns (μ ² positive area). TPH2-positive neurons were expressed as a percentage of DAPI-positive area (Figure 1B). Compared with FGF2 treatment, the addition of FGF4 followed by addition of SHH gave a higher yield of serotonin-positive neurons resulting in ∼75% of serotonin neurons.

Figure 1

Improvement in differentiation of ESC-derived serotonin neurons by mitogenic factors and SHH. (A) Schematic diagram of differentiation protocol. (B) Percentage of TPH2-positive cells in the differentiated cultures with different combination treatments with mitogenic factors (FGF2 or FGF4) and SHH (n = 4, *P < 0.05). (C) Montage of serotonin neurons immunolabeled for TPH2 (green). (a) FGF2 alone, (b) FGF2 with SHH, (c) FGF4 alone, (d) FGF4 with SHH. Scale bar 200 μm. ESC, embryonic stem cell; SHH, sonic hedgehog; TPH, tryptophan hydroxylase; FGF, fibroblast growth factor; EB, embryoid body; DAPI, 4′,6-diamidino-2-phenylindole

ESC-derived serotonin neurons have basic membrane properties

We examined whether these ESC-derived serotonin neurons have neuronal membrane properties. ESC-derived serotonin neurons had resting membrane potentials between −60 and −70 mV (n = 12). Figure 2A illustrates the striking neuronal morphology attained by a neuron that has been immunolabeled for TPH2. The electrophysiological recordings showed that ESC-derived serotonin neurons exhibit action potentials that are blocked by tetrodotoxin (TTX) (Figure 2B). Figure 2C illustrates that these cells express the GABA_A receptor/channel as is common in cultured raphe cells.¹⁶ Currents elicited by a series of depolarizing voltage steps are increased in the presence of GABA (10 μmol/L; n = 4) (Figure 2D) and reversed at hyperpolarized potentials as would be expected for the GABA_A-induced chloride current (Figure 2E). Together these data indicate that ESC-derived serotonin neurons have membrane properties that are consistent with a neuronal phenotype.

Figure 2

ESC-derived serotonin neurons elicit action currents. (A) Representative serotonin neuron. (B) Action currents in depolarizing voltage step −70 to −30 mV (n = 12). Voltage-dependent sodium channel implicated by absent action potential during TTX superfusion, recovers after washout. (C) Currents evoked by depolarizing 10 mV voltage steps. (D) Presence of GABA (n = 4). (E) Current–voltage plot of the subtracted (GABA-control). TTX, tetrodotoxin (A color version of this figure is available in the online journal)

ESC-derived serotonin neurons have high-affinity transporter and autoreceptor sites

CNS serotonin neurons have a high-affinity uptake mechanism that utilizes SERT, which is one of the major targets of the class of psychoactive drugs known as selective serotonin reuptake inhibitors (SSRIs).¹⁷ To examine whether ESC-derived serotonin neurons express SERT, [³H]-paroxetine, a common SSRI, was used in a radioligand-binding assay. ESC-derived serotonin neurons exhibited an average [³H]-paroxetine-specific binding of 87.36 fmol at a substrate concentration of 2 μmol/L. To determine the specificity of [³H]-paroxetine binding, 1 μmol/L fluoxetine, another common SSRI, was used for competition. Fluoxetine significantly displaced [³H]-paroxetine binding (Figure 3A, *P = 0.03). To further verify the specificity of [³H]-paroxetine binding, mazindol, which has a high affinity for norepinephrine transporters, was incubated along with [³H]-paroxetine. Mazindol did not displace [³H]-paroxetine binding.

Figure 3

ESC-derived serotonin neurons express functional transporter and autoreceptor sites. (A) SERT binding using [³H]-paroxetine radioligand assays. A significant reduction in [³H]-paroxetine binding in the presence of fluoxetine (n = 6, *P < 0.03) but not mazindol. (B) 5HT_1A binding using [³H]-8-OH-DPAT radioligand assays. A significant reduction in [³H]-8-OH-DPAT binding in the presence of serotonin and cold 8-OH-DPAT (n = 6, *P < 0.05). ESC, embryonic stem cell; SERT, serotonin reuptake transporter

The 5HT_1A autoreceptor is one of the major serotonin receptors that binds serotonin in the extracellular space and decreases neuronal firing and serotonin release.^18–20 Thus, it is essential that our ESC-derived serotonin neurons express functional 5HT_1A autoreceptors in order for us to proceed with psychopharmaceutical studies. ESC-derived serotonin neurons were incubated with [³H]8-OH-DPAT radioligand, 5HT_1A autoreceptor agonist, to measure the affinity of 5HT_1A autoreceptor binding. [³H]8-OH-DPAT had an average specific binding of 15.09 fmol at a substrate concentration of 2 nmol/L. The binding was specific to 5HT_1A autoreceptor as [³H]8-OH-DPAT radioligand binding was significantly displaced when incubated with either serotonin or cold 8-OH-DPAT (Figure 3B, *P < 0.05). Moreover, the Bmax was similar to that previously obtained in macaque brain.¹³

Altogether, the binding studies confirm that the ESC-derived serotonin neurons express functional proteins that define a neuron as serotonergic, and thus will be a useful in vitro primate model system.

Rhesus ESC line, ORMES-22, differentiates to serotonergic neurons with high efficiency

We used another rhesus ESC line, ORMES-22, to validate our optimized protocol's efficiency to differentiate ESC into serotonin neurons. ORMES-22 is a recently established in vitro fertilized-derived rhesus ESC line,¹¹ which was subjected to our modified protocol. Similar to rhesus ESC line 366.4, ORMES-22 differentiated into serotonin neurons as determined by TPH2 ICC (Figure 4). This suggests that our modified protocol can efficiently differentiate another rhesus ESC line, ORMES-22, into serotonin neurons.

Figure 4

Rhesus ESC ORMES-22 differentiates into serotonin neurons. Confocal photomicrograph of representative differentiated serotonin neurons immunolabeled for TPH2 (green) and DAPI (blue). Scale bars, 50 μm (top), 250 μm (bottom). TPH, tryptophan hydroxylase; DAPI, 4′,6-diamidino-2-phenylindole; ESC, embryonic stem cell (A color version of this figure is available in the online journal)

Discussion

The development of a physiologically relevant primate serotonin neural culture system that allows direct examination of cellular and molecular signaling mechanisms is essential to our understanding and development of new psychopharmacotherapies. Human and monkey ESCs differ substantially from mouse ESCs in terms of morphology and surface marker expression. The differences between human and mouse ESCs may result from fundamental differences in embryonic development between the species, or they may reflect a difference in the embryonic stage of origin of ESCs in mouse and primate. Recently, we obtained information on the genetics of the developmental cascade throughout the differentiation stages from undifferentiated ESCs to serotonin neurons.²¹ Most of our understanding in the developmental pathway leading to serotonin neurons comes from rodent models^22,23; however, fetal development in rodents and primates differs significantly. We observed similarities and differences between in vivo rodent studies and rhesus ESC-derived serotonin neurons in our microarray study. Several genes thought to be critical signals for a neural and serotonin phenotype in mice such as SHH, Pet1 (Fev), TGFβ, VEGF, and Wnt pathways were similarly expressed during in vitro differentiation of ESC-derived serotonin neurons. However, we also observed some differences in the ontogeny of Phox2b, Mash1 and Lmx1b gene expressions between our microarray study and in vivo mice studies.²⁴ In the ESC-derived serotonin neurons, Fev appeared to be the key gene for expression of other serotonin markers. Nonetheless, the differences between in vivo rodent studies and our in vitro ESC study may point to genes that are important for the organization of the serotonin system rather than major differences between rodents and primates in the expression of the serotonin phenotype.

The attractive pluripotent characteristic of stem cells also presents a significant challenge to obtain pure cultures of only one cell type. The goals of this study were to enhance our ability to differentiate rhesus ESCs into serotonergic neurons at a high yield in a predictable manner and to use these cultures to obtain physiological characterization of the ESC-derived serotonin neurons. Previously, we found that exposure of neurospheres to FGF2 helped differentiate the rhesus 366.4 ESC into serotonin neurons.⁶ Other studies using mouse ESCs showed sequential exposure of FGF4 then FGF8 and SHH led to a high yield of differentiated serotonin neurons.^7,8,25,26

Our results corroborate the mouse studies in that the sequential combination of FGF4 and SHH improved the yield of serotonin-positive neurons to ∼75% as determined by TPH2 ICC and stereology analysis. Overall, the yield of serotonin neurons and the architecture of the cultures were significantly improved. We have also confirmed that our modified protocol is efficient in differentiation of serotonergic neurons from another rhesus ESC line, ORMES-22. It may be noted that TPH2-positive ESC 366.4-derived serotonergic neurons in Figure 1C does not illustrate neurite extension as seen in Figure 4 of ESC ORMES-22-derived serotonin neurons; however, this is due to the difference in the low magnification used to obtain the montages (see scale bars in figure legends). In high magnification, both ESC 366.4 and ORMES-22-derived serotonergic neurons showed similar striking neuronal morphology with extensive neurite extensions.

Other attempts to obtain a pure monolayer ESC-derived serotonin culture included: (1) treatment with cytosine arabinoside (Ara-C), a well-known antimitotic agent, to eliminate unwanted rapidly proliferating cells while preserving non-proliferating serotonergic neurons²⁷; (2) priming with heparin and laminin to encourage migration and spreading of the neurons²⁸; (3) using different substrata such as an astrocyte cell line, extracellular matrix from astrocytes and neural cell adhesion molecule.^29,30 Although these methods showed initial promise by enhancing neuronal outgrowth and attainment of monolayers (data not shown), they also led to overgrowth of unwanted cell types and Ara-C treatment was toxic for the ESC-derived serotonin neurons.

Nonetheless, application of our modified protocol provided monolayers of ESC-derived serotonin neurons with consistent well-to-well cell numbers. This allowed us to perform functional studies and determine whether these neurons have neuronal membrane properties, as well as SERT and autoreceptor expression in a manner similar to primate CNS serotonin neurons. The electrophysiological recordings showed that ESC-derived serotonin neurons exhibit action potentials that are blocked by TTX, and that they express GABA receptors in a fashion similar to dissociated postnatal rat serotonin neurons.¹⁶ Together these data indicate that ESC-derived serotonin neurons have membrane properties that are consistent with a neuronal phenotype.

SERT is responsible for the recycle and clearance of serotonin in the brain and is the major target of SSRIs. Therefore, in order for the ESC-derived serotonin neurons to be a good in vitro model, it is essential that they express a functional transporter. Indeed, the ESC-derived serotonin neurons exhibited a high-affinity-binding site for [³H]-paroxetine that was effectively blocked with fluoxetine. In rats, [³H]-paroxetine exhibits an average Bmax of 120 fmol/mg protein, which is comparable to our SERT-binding assay.³¹ Addition of mazindol, a high-affinity norepinephrine transporter ligand, had a minor, non-significant effect in blocking [³H]-paroxetine binding. Although paroxetine has been shown to be a potent and selective inhibitor of SERT, it has recently been demonstrated to also have moderate affinity for the norepinephrine transporter (NET).³² Thus, it is possible that mazindol may exhibit low-affinity binding to serotonin transporters or that [³H]-paroxetine bound to a low number of norepinephrine transporters that were in the cultures and that were displaced by mazindol.

The 5HT_1A autoreceptor is located on the soma and dendrites of the serotonin neurons and it provides an ultra-short loop feedback mechanism thought to cause a delay in the onset of efficacy of antidepressant drugs.³³ In depressed patients, 5HT_1A autoreceptor levels are elevated and supplementation of antidepressant therapy with 5HT_1A antagonists outperforms SSRIs alone.^34,35 Thus, it is essential that our ESC-derived serotonin neurons express the 5HT_1A autoreceptor in order for us to proceed with psychopharmaceutical studies. The ESC-derived serotonin neurons exhibited a high-affinity-binding site for [³H]-8-OH-DPAT that was blocked to a significant degree with cold 8-OH-DPAT or serotonin. Moreover, the Bmax was similar to that previously obtained in macaque brain.¹³ The commonly used 5HT_1A autoreceptor agonist 8-OH-DPAT has been shown to have affinity for non-5HT_1A autoreceptors, such as serotonin uptake sites, 5HT_1D and 5HT_1B receptors, adrenoceptors and dopamine D2 receptors,^36–39 which may explain some of the [³H]-8-OH-DPAT binding that was not blocked by serotonin or cold 8-OH-DPAT in Figure 3B. Thus, it will be interesting to further examine the specificity of 5HT_1A autoreceptor binding in the presence of non-5HT_1A autoreceptor drugs such as SSRIs, ketanserin (non-5HT_1A receptor antagonist), prazosin (adrenoceptor antagonist) and raclopride (dopamine receptor antagonist). Also, there are seven distinct serotonin receptors families with at least 15 subpopulations to date; thus further studies will be needed to examine other possible receptors expressed in these ESC-derived serotonin neurons.

In conclusion, we are able to produce ESC-derived serotonin neurons with high efficiency in a predictable manner. Altogether, the electrophysiological and binding studies establish that the ESC-derived serotonin neurons express several functional proteins that define a neuron as serotonergic, and suggest that this culture system will be a useful in vitro primate model system. This will open the door for use of these cells in high throughput pharmaceutical screening for novel drugs, and for studies on the intracellular mechanism of action of antidepressants and antipsychotics. In addition, it will further the goal of transplantation into a macaque model of mental illness for restoration of serotonin function or for transport of gene vectors that promote regeneration of in situ systems.

Author contributions: YT: conception and design, experiment execution, collection and/or assembly of data, data analysis and interpretation, manuscript writing; SLI: collection and/ or assembly of data, data analysis and interpretation, manuscript writing; JSW: provision of study material; CLB: conception and design, financial support, manuscript writing, final approval of manuscript.

Footnotes

ACKNOWLEDGEMENTS

Supported by NIH grants: MH73564 and MH62677 to CLB, U54 contraceptive Center Grant HD 18185, S10RR024585, and RR000163 for the operation of Oregon National Primate Research Center (ONPRC). We are deeply grateful to the Assisted Reproductive Technology Core (Dr Shoukhrat Mitalipov) for the generation of the MEFs and embryonic stem cell cultures and to Dr Anda Cornea for her assistance in confocal microscopy.

References

Persico

, Mengual

, Moessner

, Hall

, Revay

, Sora

, Arellano

, DeFelipe

, Gimenez-Amaya

, Conciatori

, Marino

, Baldi

, Cabib

, Pascucci

, Uhl

, Murphy

, Lesch

, Keller

. Barrel pattern formation requires serotonin uptake by thalamocortical afferents, and not vesicular monoamine release. J Neurosci 2001;21:6862–73

Azmitia

. Modern views on an ancient chemical: serotonin effects on cell proliferation, maturation, and apoptosis. Brain Res Bull 2001;56:413–24

Murphy

, Lerner

, Rudnick

, Lesch

. Serotonin transporter: gene, genetic disorders, and pharmacogenetics. Mol Interv 2004;4:109–23

Bethea

, Lu

, Gundlah

, Streicher

. Diverse actions of ovarian steroids in the serotonin neural system. Front Neuroendocrinol 2002;23:41–100

Evans

, Kaufman

. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–6

Salli

, Reddy

, Salli

, Lu

, Kuo

H-C

, Pau

FK-Y

, Wolf

, Bethea

. Serotonin neurons derived from rhesus monkey embryonic stem cells: similarities to CNS serotonin neurons. Exp Neurol 2004;188:351–64

Barberi

, Klivenyi

, Calingasan

, Lee

, Kawamata

, Loonam

, Perrier

, Bruses

, Rubio

, Topf

, Tabar

, Harrison

, Beal

, Moore

, Studer

. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnol 2003;21:1200–7

Kim

, Auerbach

, Rodriguez-Gomez

, Velasco

, Gavin

, Lumelsky

, Lee

, Nguyen

, Sanchez-Pernaute

, Bankiewicz

, McKay

. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 2002;418:25–7

Kumar

, Kaushalya

, Gressens

, Maiti

, Mani

. Optimized derivation and functional characterization of 5-HT neurons from human embryonic stem cells. Stem Cells Dev 2009;18:615–27

10.

Thomson

, Kalishman

, Golos

, Durning

, Harris

, Becker

, Hearn

. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA 1995;92:7844–8

11.

Mitalipov

, Kuo

, Byrne

, Clepper

, Meisner

, Johnson

, Zeier

, Wolf

. Isolation and characterization of novel rhesus monkey embryonic stem cell lines. Stem Cells 2006;24:2177–86

12.

, Eshleman

, Janowsky

, Bethea

. Ovarian steroid regulation of serotonin reuptake transporter (SERT) binding,distribution and function in female macaques. Mol Psychiatry 2003;8:353–60

13.

, Bethea

. Ovarian steroid regulation of 5HT1A receptor binding and G protein activation in female monkeys. Neuropsychopharmacology 2002;27:12–24

14.

Walther

, Bader

. A unique central tryptophan hydroxylase isoform. Biochem Pharmacol 2003;66:1673–80

15.

Bethea

, Mirkes

, Shively

, Adams

. Steroid regulation of tryptophan hydroxylase protein in the dorsal raphe of macaques. Biol Psychiatry 2000;47:562–76

16.

Johnson

. Electrophysiological and histochemical properties of postnatal rat serotonergic neurons in dissociated cell culture. Neuroscience 1994;63:775–87

17.

Vaswani

, Linda

, Ramesh

. Role of selective serotonin reuptake inhibitors in psychiatric disorders: a comprehensive review. Prog Neuropsychopharmacol Biol Psychiatry 2003;27:85–102

18.

Sprouse

, Aghajanian

. (-)-Propanolol blocks the inhibition of serotonergic dorsal raphe cell firing by 5HT1A selective agonists. Eur J Pharmacol 1986;128:295–8

19.

Blier

, de Montigny

. Modification of 5-HT neuron properties by sustained administration of the 5-HT1A agonist gepirone: electrophysiological studies in the rat brain. Synapse 1987;1:470–80

20.

Azmitia

, Gannon

, Kheck

, Whitaker-Azmitia

. Cellular localizaiton of the 5-HT1A receptor in primate brain neurons and glial cells. Neuropsychopharmacology 1996;14:35–46

21.

Bethea

, Reddy

, Tokuyama

, Henderson

, Lima

. Protective actions of ovarian hormones in the serotonin system of macaques. Front Neuroendocrinol 2009;30:212–38

22.

Cheng

, Chen

, Luo

, Tan

, Qiu

, Johnson

, Ma

. Lmx1b, Pet-1, and Nkx2.2 coordinately specify serotonergic neurotransmitter phenotype. J Neurosci 2003;23:9961–7

23.

Hendricks

, Fyodorov

, Wegman

, Lelutiu

, Pehek

, Yamamoto

, Silver

, Weeber

, Sweatt

, Deneris

. Pet-1 ETS gene plays a critical role in 5-HT neuron development and is required for normal anxiety-like and aggressive behavior. Neuron 2003;37:233–47

24.

Bethea

, Reddy

, Pedersen

, Tokuyama

. Expression profile of differentiating serotonin neurons derived from rhesus embryonic stem cells and comparison to adult serotonin neurons. Gene Expr Patterns 2009;9:94–108

25.

Gross

, Zhuang

, Stark

, Ramboz

, Oosting

, Kirby

, Santarelli

, Beck

, Hen

. Serotonin1A receptor acts during development to establish normal anxiety-like behaviour in the adult. Nature 2002;416:396–400

26.

Lee

, Lumelsky

, Studer

, Auerbach

, McKay

. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000;18:675–9

27.

Calderon-Martinez

, Garavito

, Spinel

, Hurtado

. Schwann cell-enriched cultures from adult human peripheral nerve: a technique combining short enzymatic dissociation and treatment with cytosine arabinoside (Ara-C). J Neurosci Methods 2002;114:1–8

28.

Tarasenko

, Yu

, Jordan

, Bottenstein

, Wu

. Effect of growth factors on proliferation and phenotypic differentiation of human fetal neural stem cells. J Neurosci Res 2004;78:625–36

29.

Noble

, Fok-Seang

, Cohen

. Glia are a unique substrate for the in vitro growth of central nervous system neurons. J Neurosci 1984;4:1892–903

30.

Wujek

, Akeson

. Extracellular matrix derived from astrocytes stimulates neuritic outgrowth from PC12 cells in vitro. Brain Res 1987;431:87–97

31.

Nadgir

, Malviya

. In vivo effect of antidepressants on [3H]paroxetine binding to serotonin transporters in rat brain. Neurochem Res 2008;33:2250–6

32.

Nemeroff

, Owens

. Neuropharmacology of paroxetine. Psychopharmacol Bull 2003;37(Suppl 1):8–18

33.

Gundlah

, Hjorth

, Auerbach

. Autoreceptor antagonists enhance the effect of the reuptake inhibitor citalopram on extracellular 5-HT: this effect persists after repeated citalopram treatment. Neuropharmacology 1997;36:475–82

34.

Stockmeier

, Shapiro

, Dilley

, Kolli

, Freidman

, Rajkowska

. Increase in serotonin-1A autoreceptors in the midbrain of suicide victims with major depression-postmortem evidence for decreased serotonin activity. J Neurosci 1998;18:7394–410

35.

Artigas

, Perez

, Alvarez

. Pindolol induces a rapid improvement of depressed patients treated with serotonin reuptake inhibitors. Arch Gen Psychiatry 1994;51:248–51

36.

Kleven

, Assie

, Koek

. Pharmacological characterization of in vivo properties of putative mixed 5-HT1A agonist/5-HT(2A/2C) antagonist anxiolytics. II. Drug discrimination and behavioral observation studies in rats. J Pharmacol Exp Ther 1997;282:747–59

37.

Assie

, Koek

. [(3)H]-8-OH-DPAT binding in the rat brain raphe area: involvement of 5-HT(1A) and non-5-HT(1A) receptors. Br J Pharmacol 2000;130:1348–52

38.

Pauwels

, Colpaert

. Stereoselectivity of 8-OH-DPAT enantiomers at cloned human 5-HT1D receptor sites. Eur J Pharmacol 1996;300:137–9

39.

Brown

, MacKinnon

, McGrath

, Spedding

, Kilpatrick

. Alpha 2-adrenoceptor subtypes and imidazoline-like binding sites in the rat brain. Br J Pharmacol 1990;99:803–9

Functional characterization of rhesus embryonic stem cell-derived serotonin neurons

Abstract

Keywords

Introduction

Materials and methods

ESC culture and in vitro differentiation

Immunocytochemistry

Treatment with different mitogenic factors and SHH

Electrophysiology

SERT-binding assay

5HT1A-binding assay

Results

Improvement in viability and yield of serotonin neurons

ESC-derived serotonin neurons have basic membrane properties

ESC-derived serotonin neurons have high-affinity transporter and autoreceptor sites

Rhesus ESC line, ORMES-22, differentiates to serotonergic neurons with high efficiency

Discussion

Footnotes

ACKNOWLEDGEMENTS

References

5HT_1A-binding assay