Characterization of Induced Pluripotent Stem Cell-derived Human Serotonergic Neurons

In the brain, the serotonergic neurons located in the raphe nucleus are the unique resource of the neurotransmitter serotonin, which plays a pivotal role in the regulation of brain development and functions. Dysfunction of the serotonin system is present in many psychiatric disorders. Lack of in vitro functional human model limits the understanding of human central serotonergic system and its related diseases and clinical applications. Previously, we have developed a method generating human serotonergic neurons from induced pluripotent stem cells (iPSCs). In this study, we analyzed the features of these human iPSCs-derived serotonergic neurons both in vitro and in vivo. We found that these human serotonergic neurons are sensitive to the selective neurotoxin 5, 7-Dihydroxytryptamine (5,7-DHT) in vitro. After being transplanted into newborn mice, the cells not only expressed their typical molecular markers, but also showed the migration and projection to the host’s cerebellum, hindbrain and spinal cord. The data demonstrate that these human iPSCs-derived neurons exhibit the typical features as the serotonergic neurons in the brain, which provides a solid foundation for studying on human serotonin system and its related disorders.


INTRODUCTION
In the brain, serotonergic neurons are located in the raphe nucleus. Although there are only about 300,000 serotonergic neurons in the human brain (Chen and Condron, 2008), they innervate the whole central nervous system, with the rostral group (B5-B9 subgroups) mainly projecting to the brain and the caudal group (B1-B4 subgroups) primarily sending axons to the spinal cord (Goridis and Rohrer, 2002). Since they are the primary source and a site for re-uptake of serotonin in the brain, serotonergic neurons are essential for a series of serotonin-related neural functions (including regulating mood, affection, cognition, aggression, satiety, sleep and numerous other autonomic functions) and are a major therapeutic target for a wide spectrum of serotonin dysfunction-related psychiatric disorders (such as schizophrenia, depression, bipolar disorder, anxiety, obsessive-compulsive disorder, chronic pain syndrome and eating disorders; Bethea et al., 2002;Wichers and Maes, 2002;Donati and Rasenick, 2003). Establishment of in vitro human serotonergic neurons model system will help the understanding of their roles in brain physiology and in the development of diseases.
Deep in the brain, it is difficult to obtain human serotonergic neurons via biopsy and primary culture. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) make it possible to establish an in vitro system to investigate serotonergic neurons. Differentiation of serotonergic neurons has been attempted using mouse, monkey and human ESCs. During dopamine neurons differentiation from mouse ESCs in the presence of fibroblast growth factors (FGF2,FGF4 and/or FGF8), serotonergic neurons, based on staining for serotonin, have been observed (Lee et al., 2000;Kim et al., 2002;Barberi et al., 2003). Similarly, monkey ESCs have been differentiated to serotonergic neurons in the presence of FGF2 and/or FGF4, and the proportion of serotonin-expressing cells is reported high (Salli et al., 2004;Tokuyama et al., 2010). In a report on serotonergic neuron differentiation from human ESCs, a similar strategy (with the treatment of FGF1, FGF2, retinoic acids and serotonin) is employed for differentiation but the efficiency is very low (Kumar et al., 2009).
Recently, we have demonstrated the accurate timely regulation of WNT, SHH and FGF4 signaling pathways during the serotonergic neuron differentiation and generated an enriched population of serotonergic neurons from human ESCs and iPSCs (Lu et al., 2016). These human serotonergic neurons not only express bio-markers (TPH2, serotonin, GATA3, GATA2, FEV, LMX1B, SERT, AADC and VMAT2; Deneris and Wyler, 2012), but also show electrophysiological activities and release serotonin in response to stimuli, including FDA-approved drugs, in a dose-and time-dependent manner.
In this study, we further analyzed the features of human iPSCs-derived serotonergic neurons both in vitro and in vivo. We found that these human serotonergic neurons are sensitive to the specific neurotoxin 5,7-Dihydroxytryptamine (5,7-DHT) in vitro. After being transplanted into newborn mice, the cells not only expressed their typical molecular markers, but also showed the migration and projection to the cerebellum, hindbrain and spinal cord. The data demonstrate that these human iPSCs-derived neurons exhibit the typical features as the serotonergic neurons in the brain, which provides a solid foundation for studying on human serotonin system and its related disorders.

Immunocytochemistry
Immunostaining was performed as described previously (Lu et al., 2016). In brief, cells were fixed in 4% neutralbuffered paraformaldehyde (PFA) for 20 min at room temperature. All antibodies, sources, and dilutions are listed in Supplementary Table S1. Cell populations were counted among total cells (Hoechst labeled) using the ImageJ software. Five fields of each coverslip were chosen randomly, and three coverslips in each group were counted. Data were expressed as mean ± SEM.

Cell Transplantation and Histology
Transplantation studies were conducted following protocols approved by the Animal Care and Use Committees at the University of Wisconsin-Madison. Human iPSCsderived serotonergic progenitors (10 5 cells in 2 µl artificial cerebral spinal fluid, Harvard Apparatus) were injected 2 mm after lambda along the midline and 3 mm deep into the 4th ventricles of P1 newborn severe combined immunodeficiency (SCID)-beige (Taconic) mice. Three months after transplantation, animals were processed for histological analysis as described before Ma et al. (2012). All antibodies, sources and dilutions are listed in Supplementary  Table S1.

Statistical Analysis
Values were expressed as mean ± SEM. Differences between means were assessed by t test. A p value <0.05 was considered statistically significant.

Human iPSCs-Derived Serotonergic
Neurons Are Sensitive to 5,7-DHT In Vitro 5,7-DHT is a selective neurotoxin for serotonergic neurons. When different concentrations of 5,7-DHT were applied to the 6-week-old cultures for 3 days, human iPSCs-derived serotonergic neurons were selectively killed, which is indicated by the loss of serotonin+ cells in the culture (Figures 1A,B). In order to confirm the selective neurotoxic effect of 5,7-DHT on human iPSCs-derived serotonergic neurons, 6-hydroxydopamine (6-OHDA) was applied to the same culture system. Immunostaining data showed that 10 µM 6-OHDA could selectively kill the TH+ neurons and leave the serotonergic neurons alive in the culture (Figures 1C,D). It is noted that high concentration of 5,7-DHT (10 µM) and 6-OHDA (100 µM) showed non-specific effects and killed most of the cells in the culture (Figures 1A,C). This data demonstrates that human iPSCs-derived serotonergic neurons are sensitive to the selective neurotoxin 5,7-DHT as the serotonergic neurons in the brain. It is a very useful property for the in vitro human serotonergic neuron system, which could be applied for the selective neurotoxin screening in the future.
Human iPSCs-Derived Serotonergic Neurons Migrate and Project to the Host's Cerebellum, Hindbrain and Spinal Cord In Vivo In order to see the in vivo features of human iPSCs-derived serotonergic neurons, human iPSCs-derived serotonergic progenitors were transplanted into the 4th brain ventricle of P1 newborn mice. Three months after transplantation, human grafts, which were positive for both human specific marker STEM121 and serotonin, formed chimera with the host's tissues around the 4th brain ventricle (Figures 2A-C).  Although most of the cells were still in the grafts, a substantial number of serotonin+ human cells were found in the dorsal hindbrain area (Figure 2C), while a few serotonin+ human fibers and cell bodies were observed in the cerebellum and ventral hindbrain respectively (Figures 2D,E). More interestingly, human iPSCs-derived serotonergic neurons even sent fibers to the host's spinal cord (Figure 2F). This indicates that human iPSCs-derived serotonergic neurons could adapt to the brain environment, survive, migrate from the transplanted progenitor niches and project neurofibers to the host's central nervous system.

Transplanted Human Cells Exhibit the Typical Bio-Markers of Neurons
In order to systematically assess the features of the transplanted cells, we first detected the transplanted human cells with pan-neural markers. The majority of the transplanted human cells (in the human grafts around the 4th brain ventricle) became neurons, indicated by Tuj1 and NeuN staining (Figures 3A,B); few astrocytes, oligodendrocytes or dividing progenitors were observed, indicated by negative GFAP, MBP or Ki67 staining respectively (Figures 3C-E). This indicates that most of the human iPSCs-derived serotonergic progenitors differentiated into neurons after transplantation.

Transplanted Human Cells Exhibit the Typical Bio-Markers of Serotonergic Neurons
Transplanted cells (in the human grafts around the 4th brain ventricle) were further identified with bio-markers of serotonergic neurons. It was observed that the transplanted human cells (labeled by either STEM121 or human specific MHC1) expressed serotonin neuron specific marker serotonin and TPH2, as well as other markers for serotonergic neurons, such as GATA2, GATA3, AADC, HTR1a and VMAT2 (Figure 4). It is noted that SERT, a marker of mature and functional serotonergic neurons, was also detected in human iPSCs-derived serotonergic neurons after transplantation (Figure 4C), while it is very hard to be stained in vitro (data not shown). This data indicates that most of the transplanted cells became serotonergic neurons and the host's brain environment help them become mature and functional.

DISCUSSION
In this study, we characterized the features of human iPSCsderived serotonergic neurons both in vitro and in vivo.
As early as 17 years ago, it was reported that serotonergic neurons could be obtained from mouse ESCs (Lee et al., 2000); however, because the studies at that moment mostly focused on the effects of dopamine neurons on Parkinson's disease (Kim et al., 2002;Barberi et al., 2003), the detailed features of ESCs-derived serotonergic neurons were not well characterized. With the advance of the knowledge and techniques for neural differentiation from rhesus monkey and human ESCs, serotonergic neurons derived from primate ESCs were generated (Salli et al., 2004;Kumar et al., 2009;Tokuyama et al., 2010). Rhesus ESCs-derived serotonergic neurons not only express bio-markers for serotonergic neurons, but also exhibit functional high-affinity transporter sites, as well as high-affinity 5HT1A binding sites, which are essential targets of common psychoactive Frontiers in Cellular Neuroscience | www.frontiersin.org drugs (Salli et al., 2004;Tokuyama et al., 2010). In the human ESCs-derived serotonergic neurons, the serotonin content, the localization of serotonin vesicles and their ability to release serotonin following depolarization were characterized using a live cell serotonin imaging technique based on three-photon microscopy (Kumar et al., 2009). High efficient generation of human serotonergic neurons from both ESCs and iPSCs was reported by us recently (Lu et al., 2016). The typical bio-markers and the in vitro electrophysiological features of serotonergic neurons were assayed on these neurons; in addition, because of the enriched population of serotonergic neurons, it makes possible to detect the serotonin neurotransmitter released in the medium via ultra-performance liquid chromatographyelectrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), which facilitates this culture system to serve as a platform to validate serotonin enhancers and selective serotonin reuptake inhibitor (SSRIs). Although systematically studied in our previous work, some important features of serotonergic neurons still need to be validated on human iPSCs-derived serotonergic neurons.
Sensitivity to the selective neurotoxin 5,7-DHT is an important feature for serotonergic neurons. 5,7-DHT is a classical neurotoxin used to selectively kill serotonergic neurons and decrease concentrations of serotonin in the brain (Cairncross et al., 1977;Liu et al., 2007). Human iPSCs-derived serotonergic neurons were selectively killed by a proper dosage of 5,7-DHT, which mimics the response to 5,7-DHT as serotonergic neurons do in the brain. In our culture system, there are around 50% serotonergic neurons and 20%-25% TH+ neurons (Lu et al., 2016). This mixed culture system gives us a chance to confirm the specific neurotoxic effect of 5,7-DHT on human iPSCsderived serotonergic neurons by the application of 6-OHDA, a neurotoxic synthetic organic compound used to selectively destroy TH+ dopaminergic and noradrenergic neurons in the brain (Kelly et al., 1975). As expected, 6-OHDA selectively killed TH+ neurons but left serotonergic neurons in the culture alive. This feature demonstrates that these human iPSCs-derived cells are serotonergic neurons which are sensitive to its selective neurotoxin. In addition, it is possible to use this in vitro culture system to screen serotonergic neuron-related neurotoxins and explore the mechanisms of how the neurotoxins damage serotonergic neurons selectively.
The in vivo features of human iPSCs-derived serotonergic neurons are very important but were never reported. In order to see the characterizations of maturation, migration, projection and cell-identity of human iPSCs-derived serotonergic neurons in a brain environment, human iPSCs-derived serotonergic progenitors were transplanted into the newborn mice. Since in the brain serotonergic neurons localize in the raphe nucleus of hindbrain, a region close to the 4th brain ventricle, the progenitors were injected into the 4th brain ventricle. Three months later, human grafts were found in the wall of the host's 4th brain ventricle, and many human cells became mature neurons by expressing NeuN. Most of the transplanted cells expressed bio-markers for serotonergic neurons. More importantly, these human cells not only migrate to the areas of dorsal and ventral hindbrain, but also projected the outgrowth into cerebellum and even spinal cord. In vitro, the human iPSCs-derived serotonergic neurons expressed regional markers with the r2-3 region identities, which locate in the median raphe (Lu et al., 2016). Some of the serotonergic neurons in the median raphe project axons to the spinal cord (Bang et al., 2012;Alonso et al., 2013). Our transplantation data is consistent with the previous observation in the animal's brain. Although it is reported that serotonergic neurons in the median raphe also project ascending axons to the anterior regions, we did not found human serotonin fibers in the anterior regions of the host's brain (data not shown). It may require a longer term for observation and more careful quantification of neural fiber projection to confirm the innervation bias of human iPSCs-derived serotonergic neurons. In this study, the successful survival of the transplanted human iPSC-derived serotonergic neurons in the animal's brain is the basis for the future functional assess. Transplantation of human iPSC-derived serotonergic cells into animal models with the deficiency of brain serotonergic neurons, such as the conditional Lmx1-b knockout mice whose central serotonergic neurons are almost completely disappeared (Hodges et al., 2008), will help the understanding of the in vivo function of human iPSCs-derived serotonergic neurons. Furthermore, in the future study, synapse formation is required to be detected to confirm the ability of human iPSCsderived serotonergic neurons to form neural circuits with host's brain.
Taken together, our study demonstrates human iPSCsderived neurons exhibit the typical features of the serotonergic neurons in the brain, which provides a solid foundation for studying on human serotonin system and its related disorders.

ACKNOWLEDGMENTS
We thank Dr. Su-Chun Zhang for his support.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fncel. 2017.00131/full#supplementary-material