Use of human induced pluripotent stem cell-derived neurons as a model for Cerebral Toxoplasmosis

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Microbes and Infection xx (2016) 1e9 www.elsevier.com/locate/micinf

Original article

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Use of human induced pluripotent stem cell-derived neurons as a model for Cerebral Toxoplasmosis Naomi Tanaka a, Danah Ashour a, Edward Dratz b, Sandra Halonen a,*

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a

Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA

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Received 4 January 2016; accepted 24 March 2016 Available online ▪ ▪ ▪

Abstract Toxoplasma gondii is a ubiquitous protozoan parasite with approximately one-third of the worlds' population chronically infected. In chronically infected individuals, the parasite resides primarily in cysts within neurons in the central nervous system. The chronic infection in immunocompetent individuals has been considered to be asymptomatic but increasing evidence indicates the chronic infection can lead to neuropsychiatric disorders such as Schizophrenia, prenatal depression and suicidal thoughts. A better understanding of the mechanism(s) by which the parasite exerts effects on human behavior is limited due to lack of suitable human neuronal models. In this paper, we report the use of human neurons derived from normal cord blood CD4þ cells generated via genetic reprogramming, as an in vitro model for the study T. gondii in neurons. This culture method resulted in a relatively pure monolayer of induced human neuronal-like cells that stained positive for neuronal markers, MAP2, NFL, NFH and NeuN. These induced human neuronal-like cells (iHNs) were efficiently infected by the Prugniad strain of the parasite and supported replication of the tachyzoite stage and development of the cyst stage. Infected iHNs could be maintained through 5 days of infection, allowing for formation of large cysts. This induced human neuronal model represents a novel culture method to study both tachyzoite and bradyzoite stages of T. gondii in human neurons. © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Latent toxoplasmosis; Induced neuronal cells; Cysts

1. Introduction Toxoplasma gondii is a ubiquitous intracellular protozoan parasite with approximately one-third of the worlds' population chronically infected [1]. In chronically infected individuals, the parasite resides primarily in cysts in muscular and neural tissues [2,3]. In immunocompromised individuals, such as AIDS patients, individuals undergoing chemotherapy or transplantation recipients, the parasite can reactivate in the brain causing a severe to potentially fatal encephalitis [4]. The chronic infection in immunocompetent individuals has traditionally been considered to be asymptomatic but serological evidence indicates the chronic infection may be an etiological * Corresponding author. Tel.: þ1 406 994 5351; fax: þ1 406 994 4926. E-mail address: [email protected] (S. Halonen).

factor for development of neuropsychiatric disorders such as Schizophrenia, prenatal depression and suicidal behavior [5e11]. Furthermore, chronic Toxoplasmosis has also been associated with cryptogenic epilepsy, migraine headaches and mild cognitive effects in elderly individuals, further indicating the chronic infection exerts significant effects on neuronal activity in the central nervous system [12e16]. The mechanisms by which the parasite exerts affects on behavioral, cognitive and other neuronal functions are not well understood. A better understanding of the mechanism(s) by which the parasite exerts effects on human brain and neurological functions is limited due in part to lack of suitable human neuronal models. In the human host the parasite consists of two phases, the rapidly replicating tachyzoite stage and the slower replicating bradyzoite stage. The tachyzoite stage

http://dx.doi.org/10.1016/j.micinf.2016.03.012 1286-4579/© 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Tanaka N, et al., Use of human induced pluripotent stem cell-derived neurons as a model for Cerebral Toxoplasmosis, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.03.012

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replicates within vacuoles leading to host cell lysis while the more slowly replicating bradyzoite stage leads to formation of cysts that persist in muscle and neural tissue for the lifetime of the host [17,18]. In vivo studies have established that in the brain, neurons are the predominant host cell in which the cysts persist in the chronically infected host [19,20]. Previous in vitro studies of Toxoplasma-infected neurons have consisted primarily of mice or rat neuronal cultures composed of glial cells and neurons, and focused on differential development of tachyzoite and bradyzoite stages in glial cells vs. neurons but were not able to study development of the parasite in pure neuronal cells [21e26]. A recent study using human neurons derived from patients with brain disorders and healthy controls, reported these neurons could support growth of tachyzoite stage and cyst development and offers a potentially better in vitro model to study the effects of the parasite on human neuronal functions [27]. In this paper, we report on the use of human neurons derived from NCRM-1 cells, a neural stem cell (NSC) line derived from normal cord blood CD4þ cells generated via genetic reprogramming, as another in vitro model for the study of T. gondii in neurons. The phenotype of the NSC-derived human neurons was validated using neuronal markers, MAP2, the neurofilament heavy and light chains, NFH and NFL respectively, and NeuN. This culture method resulted in a relatively pure, high-density monolayer of neuronal-like cells. These induced human neuronal-like cells (iHNs) were efficiently infected by the Prugniad (type II) strain of the parasite and supported replication of the tachyzoite stage and cyst development. Infected iHNs could be maintained through 5 days of infection, allowing for formation of large cysts. This induced human neuronal model represents a novel and effective culture method to study effects of Toxoplasma tachyzoite and bradyzoite stages and cyst development in human neurons. 2. Materials and methods 2.1. Culture of neural stem cells (NSCs) Induced human neuronal-like cells (iHNs) were derived from the cell line, NCRM-1, obtained from NIH Center for Regenerative Medicine (http://commonfund.nih.gov/ stemcells). NCRM-1 is an induced pluripotent stem cell (ihPSC) line that was derived using the episomal vectors (Oct4, Sox2, c-Myc, Klf4, Lin28 2, and SV40 Large T antigen) as a reprogramming method. The somatic starting material was cord blood CD34þ cells. Upon this reprogramming, the NCRM-1 cells are a neural stem cell phenotype that can be passaged numerous times. In this study, NCRM-1 neural stem cells, were used to differentiate human neurons. NCRM1 cells, hereafter referred to as Neural Stem Cells (NSCs), were passaged weekly, and retained the ability to differentiate into human neurons through 20 passages. NSCs were cultured in StemPro NSC-SFM (GIBCO; Cat. No. A10509-01) consisting of KnockOUT™ DMEM/F12 media supplemented with recombinant human FGF-basic, recombinant human EGF, and StemPro Neural Supplement. The NSC-SFM media

was supplemented with GlutaMAX and AntibioticAntimycotic solution (GIBCO Cat. No.15240). This complete media called NSC-expansion media (NSC-EM) was used to expand and passage the NSC cells. NSC cells were plated onto CELLstart (GIBCO Cat no. A10142) coated 6 well plates, according to the manufacturers directions, at a cell density of 5  104 cells/cm2. NSCs were then cultured at 37  C in a humidified atmosphere of 5% CO2. NSC cells were fed with fresh NSC-EM every two days and passaged, using Accutase to dissociate, when cells were 95% confluent. NSCs maintained in culture up to 20 passages, were used in these experiments. 2.2. Differentiation of induced human neuronal-like cells (iHNs) from NSCs Neuronal differentiation was adapted from established protocols by Yan et al. [28]. Briefly, neuronal differentiation was initiated by dissociating NSCs followed by plating onto poly-L-ornithine and laminin coated dishes at a density of 4  105 cells/cm2 in NSC-EM. The next day, the spent medium was replaced with a neuronal induction medium (NIM) consisting of Neurobasal medium, MEM nonessential amino acids, GlutaMAX, B27 supplement (17504-044; GIBCO), 200 ng/ml Sonic Hedge Hog (SHH) (SRP 3156, Sigma), and 100 ng/ml recombinant human Fibroblast Growth Factor 8 (FGF8) (PHG0274; GIBCO) for 10 days, with medium changes every alternative day for 10 days. On day 10, cells were dissociated with Accutase and plated onto laminincoated four-well LabTek II chamber slides or laminin coated coverslips at a density of 2  104 cells/cm2 in a neuronal differentiation medium (NDM) consisting of Neurobasal medium, MEM nonessential amino acids, GlutaMAX, B27 supplement, 200 mm ascorbic acid, 20 ng/ml human BrainDerived Neurotrophic Factor (BDNF) (PHC7074; GIBCO), and 20 ng/ml Glial-Derived Neurotrophic Factor (GDNF) (PHC7045; GIBCO) for another 10 days, with a medium change every other day, to allow maturation of neurons. At the end of this induction/differentiation period, these cells achieved a neuronal morphology, exhibiting processes and a small cell body. These cells were characterized for neuronal markers (see Sec. 2.3 below), and found to stain positive for neuronal markers and were subsequently referred to as induced human neuronal-like cells (iHNs). 2.3. Characterization of iHNs using neuronal markers and immunofluorescence Immunocytochemical staining was used to determine the phenotype of differentiated iHNs. Differentiated iHNs were fixed with 4% paraformaldehyde at room temperature for 15 min, washed 3 with PBS and then permeabilized and blocked in a solution containing 1% Bovine Serum Albumin (BSA) and 0.05% Saponin for 30 min. Following this blocking/permeabilization step cells were incubated in primary antibodies at 4  C overnight in blocking buffer consisting of DPBS þ 1% BSA, followed by 3 washes in DPBS,

Please cite this article in press as: Tanaka N, et al., Use of human induced pluripotent stem cell-derived neurons as a model for Cerebral Toxoplasmosis, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.03.012

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incubation in Alexa Fluor 488, 594 or 647-conjugated secondary antibodies (1:200, Life Technologies) for 1 h, followed by 3 washes in DPBS. Cells were then allowed to air dry and then mounted with ProLong Gold with DAPI antifade mounting media (P-36931, Life Technologies). The following primary antibodies were used to characterize differentiated iHNs: anti-MAP-2 (Microtubule-AssociatedProtein) (1:200, AB 5622, Millipore), anti-Neurofilament heavy chain (1:200, AB 5539, Millipore); antiNeurofilament light chain (1:200; MAB 1615, Millipore), and NeuN (1:50, MAB 377, Millipore). Antibodies to the neural stem cell markers, anti-SOX2 (1:100, AB5603, Millipore) and anti-Nestin (1:100, MAB 5326, Millipore) were used to characterize the NSC cells. Fluorescent images and phase contrast images were captured on an inverted fluorescence microscope (Nikon TE2000) and analyzed using Metamorph software. The percentage of neurons was determined by staining with neuronal markers, MAP-2, NFL, NFH and NeuN, and cells positive for neuronal markers counted as iHNs. Cells were visualized with a NIKON epifluorescence microscope at 20 and 40 magnification. 2.4. Toxoplasma culture in HFF cells Tachyzoites from T. gondii PRU strain, a type II strain with a non-virulent profile in mice (LD100 ¼ 103), were maintained by in vitro culture in human foreskin fibroblasts (HFFs; ATCC® CRL-1634™) grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and antibiotic/antimycotic solution according to established protocols [29]. Parasites were harvested from infected HFF monolayers, passaged through a 27 gauge needle, centrifuged at 600  g for 10 min, counted and resuspended in NDM and used for infection of iHN cultures as described below.

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2.6. Immunofluorescence staining of Toxoplasma infected iHNs and HFFs The iHNs were inoculated with T. gondii as indicated above and at specified times, infected iHNs were fixed with 4% paraformaldehyde and processed for immunofluorescence labeling. Infected iHNs were double-labeled with anti-p30 (C86319M; Meridian Life Sciences), which reacts to the major surface protein p30 (¼SAG1) to label the parasite, and anti-MAP2 to label the neurons. For cyst experiments, infected iHNs were triply labeled with anti-p30 to label the parasites, DBA-FITC, (Dolichos Biflorans Agglutinin conjugated to fluorescein) (FL-1031; Vector Labs) to label the cyst wall and anti-MAP2 to label the neurons. For cyst experiments in HFFs, cells were double-labeled for anti-p30 and DBA-FITC and counterstained with DAPI to label host cell nuclei. Cells were visualized with a NIKON epifluorescence microscope at 20 and 40 magnification. 2.7. Quantification and statistical analysis Quantitative analyses of iHNs infected with tachyzoites or cysts were based on two independent experiments with each experiment done in triplicate. The percentage of iHNs containing tachyzoites was calculated by determining the ratio of cells double positive for MAP2 and p30, to cells that were single positive for MAP2. The percentage of parasitophorous vacuoles (PVs) in iHNs that were cyst wall positive was determined by counting the number of PVs present in MAP2 positive cells that stained with DBA-FITC. Cysts were determined by the presence of DBA-FITC immunoreactivity on the perimeter of the vacuole, using phase contrast to assess the vacuolar perimeter. Statistical analyses were performed using the student's t-test. A value of p < 0.05 was considered significant. 3. Results

2.5. Infection of iHNs and HFFs with Toxoplasma gondii iHNs were infected with freshly lysed tachyzoites harvested from HFF cells, suspended in Neuronal Differentiation Media (NDM) at an MOI of 2:1 (parasite:host cell of 2:1) with a half media change. Inoculated iHNs were incubated for 48 h at 37  C in a 5% CO2 incubator. Infected iHNs were harvested by fixation with 4% paraformaldehyde at 2 h, 24 h, and 48 h postinfection (p.i.) respectively and then processed for immunofluorescence staining as specified below. For cyst development experiments iHNs and HFFs were infected with freely lysed tachyzoites from HFF cells, at a MOI of 0.5, with a half-media change, followed by incubation for up to 5 days p.i. at 37  C in 5% CO2. Inoculated iHNs and HFFs were fixed in 4% paraformaldehyde at 24 h, 72 h and 96 h p.i. and processed for immunofluorescence staining as described below. For kinetic analysis of cyst development, iHNs were infected as described above and fixed at 12 h, 24 h, 36 h, 48 h and 96 h p.i. and processed for immunofluorescence staining as described below.

3.1. Characterization of NSC-derived induced human neuronal-like cells (iHNs) Neurons were differentiated from NSCs according to established protocols for derivation of neurons from human induced pluripotent stem cells [28,30]. Briefly, NSCs were first incubated in neuronal induction media for 10 days, followed by culture in neuronal differentiation media for an additional 10 days (Fig. 1A). By this method NSCs are transformed from cells that have flat polygonal morphology that are positive for the neural stem cell markers, SOX2 and Nestin, to cells that obtain a neuronal morphology comprised of long processes and a small cell body and are positive for neuronal marker, MAP2 (Fig. 1B and C). Between 80 and 90% of the cells stained positive for MAP2 neuronal marker after the end of the induction/differentiation period, with most of the remaining cells apoptotic or non-viable as judged by the small cell size and scant cytoplasm. Differentiation to a neuronal phenotype after the 20-day induction/differentiation period was further validated via staining with the neuronal markers, MAP2,

Please cite this article in press as: Tanaka N, et al., Use of human induced pluripotent stem cell-derived neurons as a model for Cerebral Toxoplasmosis, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.03.012

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Fig. 1. Differentiation of induced human neuronal-like cells (iHNs) from NSC. A). Schematic of differentiation of iHNs from NSC's; B). Immunofluorescence of NSCs stained with anti-SOX2 (green) and anti-Nestin (red); and C) Immunofluorescence of differentiated human neurons stained with anti-MAP2 (green) and counterstained with DAPI (blue). Abbreviations: NSC, neural stem cell; iHN, induced human neuronal-like cell; NSC-EM, neural stem cell expansion media; DIM, differentiation induction media; NDM, neuronal differentiation media; PLO ¼ poly ¼ L-ornithine; Scale bar ¼ 10 mm.

neurofilament heavy chain (NFH), neurofilament light chain (NFL) and NeuN. The majority of viable cells (over 90%) were positive for the neuronal markers, MAP2, NFH, NFL and NeuN, with NFH and NFL staining filaments in neuronal processes, MAP2 staining throughout the neuronal cell body and filaments in the neuronal processes, and NeuN expressed in the nucleus. The morphology and staining patterns were as expected for neurons, as established in the literature, and hence NSCs subjected to this differentiation scheme subsequently were called induced human neuronal-like cells (iHNs). iHNs could be maintained in neuronal differentiation medium for an additional week maintaining the differentiated neuronal markers and morphology (data not shown). 3.2. Infection of iHNs with T. gondii iHNs were infected with PRU strain of T. gondii at the end of the 20-day neuronal induction/differentiation period. iHNs were infected with parasites at an MOI of 2 and infected iHNs sampled at 2 h, 24 h and 48 h post-infection (p.i.). Cells were stained with anti-p30 to label the parasites, anti-MAP2 to label neurons, and the percentage of infected iHNs determined (Fig. 2). iHNs were readily infected with T. gondii with approximately 18% of the iHNs infected within 2 h p.i. The percentage of iHN's infected increased over the infection period with approximately 40e60% neurons infected by 24 h p.i. and 48 h p.i., respectively (Fig. 2, insert). By 24 h p.i., vacuoles typically were located near the host cell nucleus in the soma of the iHNs and vacuoles typically contained 2e4 parasites. The rate of infection increased over the course of the experiment indicating continuous invasion occurred over the 48 h time period. Many iHNs contained multiple vacuoles indicating multiple invasions per host cell had occurred over the infection period. Cultures of heavily infected iHNs, with multiple parasite vacuoles/host cell, lysed the neuronal monolayer by 72 h p.i.

3.3. Cyst development in iHNs Given the high levels of infection and multiple vacuoles/ host cell that was observed in initial experiments with iHNs, subsequent experiments were done to analyze cyst development in iHNs using a low multiplicity of infection (MOI ¼ 0.5) to obtain lower rates of neuronal infection, in order to allow time for cyst development to occur. Under these infection conditions, the percentage of infected iHNs was low (less than 10%) with infected iHNs typically containing only 1 vacuole/host cell and infected iHNs were able to be followed for up to 5 days post-infection. Infected iHNs were analyzed at 24 h, 72 h and 96 h p.i. and stained with DBA-FITC, which stains the cyst wall, anti-p30 to identify parasites and counter-stained with anti-MAP2 to identify neurons, and the percentage of parasitophorous vacuoles (PVs) in iHNs that were cyst wall positive determined. At 24 h p.i., approximately 23% of PVs expressed cyst wall antigen, with the percentage of PVs expressing cyst wall antigen increasing to 69% by 72 h p.i. and to almost 90% by 96 h p.i. (Fig. 3). For comparison, human foreskin fibroblasts (HFFs) were similarly infected with PRU strain. HFFs contained about half as many PVs expressing cyst wall antigen as neurons at 24 h p.i. (11% vs. 23% respectively) and the percentage of cyst wallþ PVs decreased to less than 1% over the infection period. The neuronal monolayer was still intact at 96 h p.i. and large cysts (20e30 mm) were evident (Fig. 4A, cysts indicated by white asterisks). Vacuoles containing single parasites were also present at 96 h p.i. indicating re-invasion had begun to occur (Fig. 4A, arrow). Most of the small vacuoles, containing 1e2 parasites, also stained positive for the cyst wall at 96 h p.i. (Fig. 4B; white arrows). The cyst wall positive vacuoles containing 1 parasite, indicates recent parasite invasions after 96 h p.i. incubation in neurons, are initiated as cysts.

Please cite this article in press as: Tanaka N, et al., Use of human induced pluripotent stem cell-derived neurons as a model for Cerebral Toxoplasmosis, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.03.012

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Fig. 2. Infection of induced human neuronal-like cells (iHNs) with Toxoplasma gondii. The percentage of tachyzoite-infected iHNs infected with PRU strain at 2 h, 24 h and 48 h p.i., at an MOI of 2.0. Infected iHNs were determined via immunofluorescence using anti-p30 to label the parasites and iHNs identified via labeling with anti-MAP2. Insert, showing the percentage of infected iHNs with standard error at 2 h, 24 h and 48 h p.i.

Fig. 3. Development of cysts in induced human neuronal-like cells (iHNs) vs. Human Foreskin Fibrobasts (HFFs). The percentage of parasitophorous vacuoles (PVs) that stained positive for cyst wall in iHNs as compared to HFFs, at 24 h, 72 h and 96 h post-infection (p.i.). Cysts were as determined by staining with Dolichos biflorans-FITC, and parasitophorous vacuoles determined via staining for anti-p30 to visualize the parasites. The percentage of cyst wall positive PVs in iHNs vs. HFFs was statistically significantly greater at 24 h, 48 h and 96 h p.i. (*indicates p < .05; ** indicates p < .01). Results are representative of two independent experiments. Hashed bars are iHNs; black bars are HFFs.

Please cite this article in press as: Tanaka N, et al., Use of human induced pluripotent stem cell-derived neurons as a model for Cerebral Toxoplasmosis, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.03.012

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Fig. 4. Immunofluorescence of cysts generated in induced Human Neuronal elike Cells (iHNs). A). Phase contrast image with fluorescence overlay showing 3 large cysts (green; white asterisks) and some small parasite vacuoles (red; white arrow) in HNs at 4 days p.i.; B). DBA-FITC staining of cysts in iHNs at 96 h p.i., white arrows indicate small cysts containing 1e2 parasites, white asterisks indicate larger cysts; CeE). Immunofluorescence of cysts (green) and parasites (red) of infected iHN at 12 h p.i., 24 h p.i. and 96 h p.i., respectively; green ¼ DBA-FITC; red ¼ anti-p30; blue ¼ DAPI; Scale bar for A ¼ 30 mm; Scale bar for B ¼ 15 mm; Scale bar for CeE ¼ 10 mm.

3.4. Kinetic analysis of cyst growth and development in iHNs To study the development of cysts in iHNs, a kinetic analysis of cyst development in iHNs was done via analyzing infected neurons at 12 h, 24 h, 36 h, 48 h, and 96 h p.i., staining the infected iHNs with DBA-FITC, to label the cyst wall, anti-p30, to label the parasite, anti-MAP2 to identify iHNs and DAPI to label host cell and parasite nuclei. Cyst wall staining of vacuoles was first evident at 12 h p.i., with vacuoles of single parasites beginning to display circumferential staining around the vacuole, and the intracellular parasite staining positive with p30 (Fig. 4C). By 24 h p.i., complete circumferential staining of the cyst wall was evident (Fig. 4D). While parasite staining with p30 staining was strong at 12 h p.i., by 24 h p.i. parasites within cyst wall positive vacuoles only faintly stained with p30, indicative of transition from the tachyzoite to the bradyzoite stage. The intensity of cyst wall staining and diameter of cysts increased throughout the incubation period, such that by 96 h p.i. large cysts, 20e40 mm in diameter, exhibiting definite staining of the cyst wall were formed (Fig. 4E). By 96 h p.i., p30 staining of parasites within the large cysts was either absent or minimal, with staining persisting only in a portion of the cyst and appearing to

localize to the internal matrix of the cyst as opposed to the parasite surface, as illustrated in Fig. 4E. In addition by 96 h p.i., a small percentage of the cyst wall positive vacuoles (10e15%) did not stain for p30. Most of the cyst wall positive vacuoles that were negative for p30 were of large size, perhaps indicating more mature cysts. 4. Discussion In this paper we report on the use of human induced pluripotent stem cells (hiPSC) to generate human neurons as an in vitro model for the culture of T. gondii. Neurons generated via these methods produced relatively pure populations of human neuronal-like cells that are easily invaded by the parasite and support replication of tachyzoite stage. Additionally, in these induced human neuronal-like cells (iHNs) a high proportion of the parasite vacuoles spontaneously formed cysts, a phenomenon not observed in fibroblasts. These iHNs supported development of cysts through 5 days in culture generating large cysts, 20e40 mm diameter with a well-defined cyst wall. Thus this induced human neuron culture provides an effective in vitro model to study Toxoplasma tachyzoite stage and development of cysts in human neurons.

Please cite this article in press as: Tanaka N, et al., Use of human induced pluripotent stem cell-derived neurons as a model for Cerebral Toxoplasmosis, Microbes and Infection (2016), http://dx.doi.org/10.1016/j.micinf.2016.03.012

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In this study using iHNs as host cell for T. gondii, a very high proportion of the parasite vacuoles spontaneously formed cysts. Furthermore in iHNs, cyst wall antigen was first evident around the vacuoles as early as 12 h p.i., with prominent circumferential staining of the vacuole evident by 24 h p.i., with a concomitant decrease in p30 staining, indicating in a neuronal host cell, tachyzoite to bradyzoite differentiation conversion to cysts begins soon after invasion. This phenomenon of spontaneous cyst formation is not observed in nonneuronal cells such as fibroblasts or other neuronal cells such as astrocytes, where stress conditions such as pH, heat shock proteins, or immune factors such as nitric oxide or cytokines, induce bradyzoite conversion [23,31e33]. The recent study by Passeri et al. [27] using human neurons derived from Schizophrenia patients and healthy controls, also found infection of human neuron host cells lead to spontaneous cyst development. Several older studies using murine or rodent mixed neuronal cultures found the number of bradyzoites increased in time in culture while the number of tachyzoites decreased, also suggesting a neuronal environment was conducive to bradyzoite differentiation, although in these studies the host cell supporting the cyst stage could not be determined [24,25]. Collectively, these observations from the literature and the results from this study and Passeri's study, indicate the neuronal host cell environment, independent of stress or host immune factors, induces bradyzoite conversion and cyst formation. Further study is required to elucidate the host cell factors involved in spontaneous bradyzoite stage conversion and cyst formation in neurons. Studies investigating stage conversion from tachyzoite to bradyzoite stage have found that while the tachyzoite plays a role in regulating the initiation of bradyzoite differentiation, induction of bradyzoite gene expression is known to be influenced by the host cell environment [34e36]. The molecule CDA-1 (cell division auto-antigen 1) has been identified as a host cell factor that can induce bradyzoite differentiation and cyst formation [35,36]. For example, overexpression of CDA-1 in HFFs induced bradyzoite gene expression and increased tissue cyst formation [35]. CDA-1 arrests the cell cycle of HFF, suggesting a link between withdrawal from the host cell cycle and bradyzoite differentiation. Spontaneous stage conversion and cyst formation has also been reported to occur in primary and skeletal muscle cells [33,37,38]. Recently, in murine skeletal muscle cells (SkMCs) it was shown that withdrawal from the cell cycle via a murine analog of CDA-1 (Tspyl2), triggered bradyzoite conversion and cyst formation [39]. Neurons, like muscle cells, are permanently withdrawn from the cell cycle, suggesting cell cycle withdrawal may also play be the physiological trigger involved in bradyzoite differentiation in neurons. Experiments are currently in progress to further investigate this hypothesis in our human neuronal culture. This induced human neuronal model presented in this study is similar to the study by Passeri et al. [27]. However in the induced human model by Passeri et al., human neurons were derived from fibroblasts, some of which remained undifferentiated in the human neuron culture and supported tachyzoite

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growth. In the human neuronal model presented in this paper, the culture began as NSCs and almost all cells differentiated to iHNs and thus there were few undifferentiated cells that would support tachyzoite growth during the incubation period. This could explain the high proportion of vacuoles that converted to cysts and the large size of cysts that developed by 4e5 days in the iHNs in our culture system. Despite these differences, both of these induced human neuronal models offer distinct advantages over other neuronal models using neuroblastoma cell lines, primary neurons derived from mice or rats, or human neurons from retina or aborted fetal tissue, that have previously been used [23,40e42]. First, induced neurons possess differentiated cell traits such as the ability to produce action potentials, to secrete neurotransmitters and to form synapses, features that may not be present or functional, in neuronal cell lines. Thus functional aspects of the hosteparasite interactions such as the effect of infection on the neuronal synapse or investigations into mechanisms by which infected neurons may become functionally silenced, as reports from in vivo studies suggest, could be addressed with this model [43,44]. Secondly, human neurons are more relevant than neurons derived from other species such as mice or rats. Thirdly, the induced human neuron model presented in this paper, offers the ability to generate unlimited, relatively pure populations of human neurons for experimentation. Finally, the use of induced human neurons derived via genetic reprogramming of somatic cells, affords the possibility of deriving neurons from patients with Schizophrenia or other neuronal disorders, for use in studies addressing the possible etiological role of Toxoplasma infection in different neurological disorders. Thus the use of the human neurons derived from induced human neuronal models, as presented here in this report and in the model by Passeri et al. [27], provide much needed in vitro neuronal models to study Toxoplasma bradyzoite stage and cyst development, the clinically relevant stages of the parasite in the chronically infected host. Previous work using the method adopted in this paper of deriving human neurons from hiPSCs have analyzed these induced human neurons via gene expression analysis, validating the phenotypic characteristics of these neurons, characterized the maturity of these induced human neurons as judged by such characteristics as neurite length, and determined that these induced human neurons can generate neuronal synapses when cultured in the presence of astrocytes [30,45,46], These induced neurons also display multipotentiality and have been shown to differentiate into other neuronal subtypes such as dopaminergic, GABAminergic, motor neurons and astrocytes when cultured with specific transcription factors [28,30]. These induced human neurons can also be adapted to 96-well formats for high throughput analysis and drug screens [45,46]. Thus this induced human neuronal model is amenable to experimentation including the capability of expansion to high throughput studies and affords many advantages as an in vitro model for the study of Cerebral Toxoplasmosis. The hiPSC-derived human neuron model presented in this report provides an effective human neuron model for the

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growth of the tachyzoite stage, bradyzoite stage and cyst development in neuron host cell. This human neuronal model affords questions of host cell/parasite interactions in neurons and specifically bradyzoite/neuron cell interactions and cyst development, in neurons to be addressed. Finally, the use of induced human neurons offers the possibility of generating human neurons from patient-specific cells and the possibility of disease modeling using patient derived human neurons, to address the impact of T. gondii in various neurological disorders. Conflict of interest The authors declare that they have not competing interests for this specific manuscript. Acknowledgments This work was supported by Manco Gulf Group (MANGUL) grant W5044 and NIH-NIAID grant 8 P20 GM1033394-05. References [1] Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol 2000;30(12e13):1217e58. [2] Ferguson DJ, Hutchison WM. An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice. Parasitol Res 1987;73(6):483e91. [3] Dubey JP. Bradyzoite-induced murine toxoplasmosis: stage conversion, pathogenesis, and tissue cyst formation in mice fed bradyzoites of different strains of Toxoplasma gondii. J Eukaryot Microbiol 1997;44(6): 592e602. [4] Luft BJ, Remington JS. Toxoplasmic encephalitis in AIDS. Clin Infect Dis Off Publ Infect Dis Soc Am 1992;15(2):211e22. [5] Okusaga O, et al. Toxoplasma gondii antibody titers and history of suicide attempts in patients with schizophrenia. Schizophr Res 2011; 133(1e3):150e5. [6] Groer MW, et al. Prenatal depression and anxiety in Toxoplasma gondiipositive women. Am J Obstet Gynecol 2011;204(5). 433 e1e7. [7] Sutterland AL, et al. Beyond the association. Toxoplasma gondii in schizophrenia, bipolar disorder, and addiction: systematic review and meta-analysis. Acta Psychiatr Scand 2015;132(3):161e79. [8] Torrey EF, Bartko JJ, Yolken RH. Toxoplasma gondii and other risk factors for schizophrenia: an update. Schizophr Bull 2012;38(3):642e7. [9] Pedersen MG, et al. Toxoplasma gondii infection and self-directed violence in mothers. Arch Gen Psychiatry 2012;69(11):1123e30. [10] Yagmur F, et al. May Toxoplasma gondii increase suicide attemptpreliminary results in Turkish subjects? Forensic Sci Int 2010; 199(1e3):15e7. [11] Pedersen MG, et al. Toxoplasma infection and later development of schizophrenia in mothers. Am J Psychiatry 2011;168(8):814e21. [12] Beste C, et al. Latent Toxoplasma gondii infection leads to deficits in goal-directed behavior in healthy elderly. Neurobiol Aging 2014;35(5): 1037e44. [13] Yazar S, et al. Investigation of probable relationship between Toxoplasma gondii and cryptogenic epilepsy. Seizure J Br Epilepsy Assoc 2003; 12(2):107e9. [14] Palmer BS. Meta-analysis of three case controlled studies and an ecological study into the link between cryptogenic epilepsy and chronic toxoplasmosis infection. Seizure J Br Epilepsy Assoc 2007;16(8): 657e63.

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