BIREY FIKRI (US)
WO2018234491A1 | 2018-12-27 |
US20180119098A1 | 2018-05-03 | |||
US20160348101A1 | 2016-12-01 | |||
US20110145179A1 | 2011-06-16 |
YUANWEI YAN; JULIE BEJOY; MARK MARZANO; YAN LI: "The Use of Pluripotent Stem Cell -Derived Organoids to Study Extracellular Matrix Development during Neural Degeneration", CELLS, vol. 8, no. 3, 14 March 2019 (2019-03-14), pages 1 - 30, XP055641500, DOI: 10.3390/cells8030242
THAT WHICH IS CLAIMED IS: 1. A method for producing a human raphe nuclei-like spheroid or organoid (hRNS) in vitro, the method comprising: (a) inducing a human pluripotent stem cell in 3D suspension culture to a neural fate to generate a neural spheroid; (b) differentiating the neural spheroid into a hRNS; and (c) maintaining the hRNS in neural medium, such that the hRNS comprises serotonergic neurons. 2. The method of claim 1, wherein the human pluripotent stem cell is an induced human pluripotent stem cell. 3. The method of claim 1, wherein inducing the human pluripotent stem cell in suspension culture to the neural fate in step (a) comprises culturing in a medium comprising an inhibitor of bone morphogenetic protein (BMP) and an inhibitor of transforming growth factor ȕ (TGFb), and wherein the method comprises supplementing the medium with an inhibitor of GSK- 3. 4. The method of claim 3, wherein the inhibitor of BMP is dorsomorphin and the inhibitor of TGFb is SB-431542. 5. The method of claim 3 or claim 4, wherein the inhibitor of GSK-3 is CHIR99021. 6. The method of claim 3, wherein culturing in the medium comprising the inhibitor of BMP and inhibitor of TGFb is for a period of from 2 to 10 days. 7. The method of claim 6, wherein the medium is supplemented with the inhibitor of GSK-3 after a period of 1 to 2 days such that the medium comprises the inhibitor of BMP, inhibitor of TGFb and inhibitor of GSK-3, optionally wherein culturing in the medium comprising the inhibitor of BMP, inhibitor of TGFb and inhibitor of GSK-3 is for a period of from 4 to 8 days. 8. The method of claim 7, wherein culturing in the medium comprising the inhibitor of BMP, inhibitor of TGFb and inhibitor of GSK-3 comprises: (1) culturing in a medium comprising the inhibitor of BMP, the inhibitor of TGFb, and inhibitor of GSK-3 for a period of 2 to 5 days, wherein the inhibitor of TGFb is present at a concentration of between 5 mM to 20 mM; and subsequently (2) culturing in a medium comprising the inhibitor of BMP, the inhibitor of TGFb, and inhibitor of GSK-3 for a period of 2 to 5 days, wherein the inhibitor of TGFb is present at a concentration of between 1 mM to 5 mM. 9. The method of claim 1, wherein the suspension culture of step (a) is feeder layer free. 10. The method of claim 1, wherein differentiating the neural spheroid into the hRNS of step (b) comprises culturing the neural spheroid in suspension culture in neural medium comprising an inhibitor of GSK-3 and a sonic hedgehog pathway agonist, and wherein the method comprises supplementing the neural medium with FGF4. 11. The method of claim 10, wherein the inhibitor of GSK-3 is CHIR99021. 12. The method of claim 10 or claim 11, wherein the sonic hedgehog (SHH) pathway agonist is smoothened agonist (SAG). 13. The method of claim 10, wherein the neural spheroid is cultured in neural medium comprising the inhibitor of GSK-3 and a SHH pathway agonist for a period of 1 to 3 weeks, and wherein the neural medium is supplemented with FGF4 after a period of 2 to 10 days. 14. The method of claim 1, wherein step (b) further comprises supplementing the neural medium with at least one of the compounds selected from the group consisting of: brain- derived neurotrophic factor (BDNF), NT3, L-Ascorbic Acid 2-phosphate Trisodium Salt (AA), N6, 2’-O-Dibutyryladenosine 3’, 5’ -cyclic monophosphate sodium salt (cAMP), cis-4, 7, 10, 13, 16, 19- Docosahexaenoic acid (DHA), and DAPT, optionally wherein the neural medium is supplemented with the at least one compound after a period of 1 to 3 weeks. 15. The method of claim 1, wherein differentiating the neural spheroid into the hRNS of step (b) comprises: (1) culturing the neural spheroid in suspension culture in neural medium comprising an inhibitor of GSK-3 and a sonic hedgehog pathway agonist for a period of 2 to 10 days; (2) supplementing the neural medium with FGF4; and (3) culturing the neural spheroid in suspension culture in neural medium comprising an inhibitor of GSK-3, sonic hedgehog pathway agonist and FGF4 for a period of 1 to 3 weeks. 16. The method of claim 15, further comprising following step (3): (4) culturing the neural spheroid in suspension culture for a period of 2 to 10 days in neural medium comprising FGF4 and at least one of the compounds selected from the group consisting of: brain-derived neurotrophic factor (BDNF), NT3, L-Ascorbic Acid 2-phosphate Trisodium Salt (AA), N6, 2’-O-Dibutyryladenosine 3’, 5’ -cyclic monophosphate sodium salt (cAMP), cis-4, 7, 10, 13, 16, 19- Docosahexaenoic acid (DHA), and DAPT; and (5) culturing the neural spheroid in suspension culture for at least 1 week in neural medium comprising the at least one compound in the absence of FGF4. 17. The method of claim 1, wherein maintaining the hRNS in step (c) is carried out in neural medium in the absence of growth factors. 18. The method of claim 1, wherein maintaining the hRNS in step (c) is for at least 1 week. 19. The method of claim 1, wherein cells of the hRNS comprise at least one allele associated with a neurologic or psychiatric disorder. 20. The method of claim 17, wherein the neurologic or psychiatric disorder is selected from the group consisting of: schizophrenia, affective disorder and autism spectrum disorder (ASD), optionally wherein the affective disorder is major depressive disorder, bipolar disorder or an anxiety disorder. 21. A human raphe nuclei spheroid (hRNS) comprising human serotonergic neurons, wherein the hRNS is capable of being maintained in suspension culture without adhering to a surface for at least 1 month. 22. A human raphe nuclei spheroid (hRNS) obtained by the method of claim 1. 23. A method for producing a cortico-raphe nuclei assembloid (hCS-hRNS) in vitro, the method comprising: (i) (a) inducing a first human pluripotent stem cell in suspension culture to a neural fate to provide a first neural spheroid; (b) differentiating the first neural spheroid into a human raphe nuclei spheroid (hRNS), (ii) (a) inducing a human pluripotent stem cell in a second suspension culture to a neural fate to derive a second neural spheroid; (b) differentiating the second neural spheroid into a cortical spheroid (hCS), and (iii) culturing the hRNS and hCS under conditions permissive for cell fusion in neural medium, such that the cortico-raphe assembloid comprises human serotonergic neurons with projections between the hRNS and hCS, and neurons from the hCS projecting into hRNS. 24. The method of claim 23, wherein human neurons form bidirectional projections between the hRNS and hCS. 25. The method of claim 23, wherein the first and/or second human pluripotent stem cell is an induced human pluripotent stem cell. 26. The method of claim 23, wherein inducing the first human pluripotent stem cell in suspension culture to the neural fate in step (i)(a) comprises culturing in a medium comprising an inhibitor of bone morphogenetic protein (BMP) and an inhibitor of transforming growth factor ȕ (TGFb), and wherein the method comprises supplementing the medium with an inhibitor of GSK- 3. 27. The method of claim 23, wherein the inhibitor of BMP is dorsomorphin and the inhibitor of TGFb is SB-431542. 28. The method of claim 23 - 26, wherein the inhibitor of GSK-3 is CHIR99021. 29. The method of claim 23, wherein culturing in the medium comprising the inhibitor of BMP and inhibitor of TGFb is for a period of from 2 to 10 days. 30. The method of claim 29, wherein the medium is supplemented with the inhibitor of GSK-3 after a period of 1 to 2 days such that the medium comprises the inhibitor of BMP, inhibitor of TGFb and inhibitor of GSK-3, optionally wherein culturing in the medium comprising the inhibitor of BMP, inhibitor of TGFb and inhibitor of GSK-3 is for a period of from 4 to 8 days. 31. The method of claim 30, wherein culturing in the medium comprising the inhibitor of BMP, inhibitor of TGFb and inhibitor of GSK-3 comprises: (1) culturing in a medium comprising the inhibitor of BMP, the inhibitor of TGFb, and inhibitor of GSK-3 for a period of 2 to 5 days, wherein the inhibitor of TGFb is present at a concentration of between 5 mM to 20 mM; and subsequently (2) culturing in a medium comprising the inhibitor of BMP, the inhibitor of TGFb, and inhibitor of GSK-3 for a period of 2 to 5 days, wherein the inhibitor of TGFb is present at a concentration of between 1 mM to 5 mM. 32. The method of claim 23, wherein differentiating the first neural spheroid into the hRNS of step (i)(b) comprises culturing the neural spheroid in suspension culture in neural medium comprising an inhibitor of GSK-3 and a SHH pathway agonist, and wherein the method comprises supplementing the neural medium with FGF4. 33. The method of claim 32, wherein the inhibitor of GSK-3 is CHIR99021. 34. The method of claim 32-33, wherein the SHH pathway agonist is smoothened agonist (SAG). 35. The method of claim 32, wherein the neural spheroid is cultured in neural medium comprising the inhibitor of GSK-3 and a sonic hedgehog pathway agonist for a period of 1 to 3 weeks, and wherein the neural medium is supplemented with FGF4 after a period of 2 to 10 days. 36. The method of claim 23, wherein step (i)(b) further comprises supplementing the neural medium with at least one of the compounds selected from the group consisting of: brain- derived neurotrophic factor (BDNF), NT-3, L-Ascorbic Acid 2-phosphate Trisodium Salt (AA), N6, 2’-O-Dibutyryladenosine 3’, 5’ -cyclic monophosphate sodium salt (cAMP), cis-4, 7, 10, 13, 16, 19- Docosahexaenoic acid (DHA), and DAPT, optionally wherein the neural medium is supplemented with the at least one compound after a period of 1 to 3 weeks. 37. The method of claim 23, wherein differentiating the first neural spheroid into the hRNS of step (i)(b) comprises: (1) culturing the neural spheroid in suspension culture in neural medium comprising an inhibitor of GSK-3 and a sonic hedgehog pathway agonist for a period of 2 to 10 days; (2) supplementing the neural medium with FGF4; and (3) culturing the neural spheroid in suspension culture in neural medium comprising an inhibitor of GSK-3, sonic hedgehog pathway agonist and FGF4 for a period of 1 to 3 weeks. 38. The method of claim 37, further comprising following step (3): (4) culturing the neural spheroid in suspension culture for a period of 2 to 10 days in neural medium comprising FGF4 and at least one of the compounds selected from the group consisting of: brain-derived neurotrophic factor (BDNF), NT3, L-Ascorbic Acid 2-phosphate Trisodium Salt (AA), N6, 2’-O-Dibutyryladenosine 3’, 5’ -cyclic monophosphate sodium salt (cAMP), cis-4, 7, 10, 13, 16, 19- Docosahexaenoic acid (DHA), and DAPT;and (5) culturing the neural spheroid in suspension culture for at least 1 week in neural medium comprising the at least one compound in the absence of FGF4. 39. The method of claim 23, wherein step (iii) comprises culturing the hRNS and hCS are cultured under conditions permissive for cell fusion for at least 3 days. 40. The method of claim 23, wherein step (iii) comprises culturing the hRNS and hCS in direct physical contact to each other. 41. The method of claim 23, wherein the suspension culture of step (i)(a) and/or step (ii)(a) is feeder layer free culture condition. 42. The method of claim 23, wherein cells of the cortico-raphe nuclei assembloid comprise at least one allele or genetic event associated with a neurologic or psychiatric disorder. 43. The method of claim 23, wherein either the first or second human pluripotent stem cell comprises at least one allele or genetic event associated with a neurologic or psychiatric disorder. 44. The method of claim 42-43, wherein the neurologic or psychiatric disorder is selected from the group consisting of: schizophrenia, affective disorder and autism spectrum disorder (ASD), optionally wherein the affective disorder is major depressive disorder, bipolar disorder or an anxiety disorder. 45. A cortico-raphe nuclei assembloid (hCS-hRNS) comprising a raphe nuclei spheroid (hRNS) fused to a cortical spheroid (hCS), wherein the hCS-hRNS comprises human serotonergic neurons projecting between the hRNS and hCS and forming a human neuromodulatory circuit, and wherein the hCS-hRNS is capable of being maintained in suspension culture without adhering to a surface for at least 4 weeks. 46. A cortico-raphe nuclei assembloid produced by the method of any of claims 23-44. 47. A method of determining the effect of a candidate agent on serotonergic modulation on cortical neural circuits, the method comprising: contacting the candidate agent with the cortico-raphe nuclei assembloid (hCS-hRNS) of claim 45-46; and determining the effect of the candidate agent on the ability of serotonergic neurons to modulate function of cortical neural circuits, optionally wherein cells of the hCS-hRNS comprise at least one allele or genetic event associated with a neurologic or psychiatric disorder, further optionally wherein the neurologic or psychiatric disorder is selected from the group consisting of: schizophrenia, affective disorder and autism spectrum disorder (ASD). 48. The method of claim 47, wherein the candidate agent is a selective serotonin reuptake inhibitor (SSRI). |
[0059] As demonstrated in the examples, the combined use of an inhibitor of GSK-3, a sonic hedgehog pathway agonist, and FGF4 results in the formation of hRNS with high levels of markers indicative of the human raphe nuclei, e.g. at least 2 weeks after the suspension culture of hiPS cells was induced to a neural fate. For example, the hRNS may have high levels of transcription factors that drive caudal midbrain/hindbrain development such as NKX6-1, NKX2-2, OLIG2, GATA2, GATA3, LMX1B, FOXA2, EN1 but low levels of forebrain markers such as FOXG1. Methods for determining levels of transcription factor expression include RT-qPCR as further described in examples. In some embodiments, the methods disclosed herein further comprise determining whether the hRNS express transcription factors that drive caudal midbrain/hindbrain development. A hRNS having high or low levels of a transcription factor may have a significantly higher or lower level of gene expression when compared to gene expression in a non-raphe nuclei spheroid, e.g. a cortical spheroid (hCS), when calculated using a standard statistical test.
[0060] To promote differentiation of neural progenitors into neurons, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks after transferring the neural spheroids to the neural medium, the neural medium is changed to replace the inhibitor of GSK-3, a sonic hedgehog pathway agonist with an effective dose of BDNF and NT3. The growth factors can be provided at a concentration for each of at least about 0.5 ng/ml, at least about 1 ng/ml, at least about 5 ng/ml, at least about 10 ng/ml, up to about 500 ng/ml, up to about 250 ng/ml, up to about 100 ng/ml, up to about 20 ng/ml, or about 10 ng/ml.
[0061] The neural medium at this stage may be optionally supplemented with an effective dose of one or more of the following agents that promote neuronal activity in general: a gamma secretase inhibitor, e.g. DART at a concentration of from about 1 to 25 mM, about 2 to 10 mM, and may be around about 2.5 mM; L-ascorbic acid at a concentration of from about 10 to 500 nM, from about 50 to 250 nM, and may be about 200 nM; cAMP at a concentration of from about 10 to 500 nM, from about 50 to 150 nM, and may be about 100 nM; and Docosahexaenoic acid (DHA) at a concentration of from about 1 mM to 100 mM, from about 5 mM to about 50 mM, from 5 mM to 25 mM, or may be about 10 mM. In some embodiments, the neural medium comprises an effective dose of BDNF, NT3, a gamma secretase inhibitor, L-ascorbic acid, cAMP and DHA.
[0062] To promote differentiation of neural progenitors into neurons, the neural spheroids may be cultured in the neural medium comprising the factors listed above for at least about 1 week, at least about 2 weeks, at least about 3 weeks, up to about 6 weeks, up to about 5 weeks, up about 4 weeks, between about 1 and about 3 weeks, or about 2 weeks. The neural medium may further comprise FGF4 for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, up to about 10 days, up to about 8 days, up to about 6 days, between about 2 to about 10 days, or about 5 days. [0063] For example, the step of promoting differentiation of neural progenitors into neurons may comprise: (4) culturing the neural spheroid in suspension culture for a period of 2 to 10 days in neural medium comprising FGF4 and at least one of the compounds selected from the group consisting of: brain-derived neurotrophic factor (BDNF), NT-3, L-Ascorbic Acid 2-phosphate Trisodium Salt (AA), N6, 2’-O-Dibutyryladenosine 3’, 5’ -cyclic monophosphate sodium salt (cAMP), cis-4, 7, 10, 13, 16, 19- Docosahexaenoic acid (DHA), and DAPT; and (5) culturing the neural spheroid in suspension culture for at least 1 week in neural medium comprising the at least one compound in the absence of FGF4. [0064] About 1 week, 2 weeks, 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks after transferring the neural spheroids to the neural medium, the spheroids can be maintained for extended periods of time in neural medium, e.g. for periods of 1 week, 2 weeks, 3 weeks, 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer. In some embodiments, the spheroids are maintained for a period of 3 months or longer. The spheroids may be maintained in a neural medium in the absence of growth factors. [0065] The hRNS comprises functional serotonergic neurons. As mentioned above, a large proportion of neurons that originate in the human raphe nuclei are serotonergic neurons which project into multiple locations of the human central nervous system, including areas of the human cortex. The presence of serotonergic neurons can be detected for example by using methods such as immunohistochemistry to determine expression of serotonin, enzymes involved in the serotonin pathway, such as tryptophan 5-hydroxylase 2 (TPH2), and/or markers of mature serotonin neurons such as vesicular monoamine transporter 2 (VMAT2) and serotonin reuptake transporter (SERT). The functionality of the neurons can be determined by monitoring neuronal activity, e.g. by imaging Ca 2+ activity. [0066] Human cortical spheroids. hCS may be generated by the methods previously described, for example in Pasca et al. (2015) Nat. Methods 12(7):671-678, entitled “Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture”, herein specifically incorporated by reference. [0067] For example, a suspension culture of hiPS cells is cultured to provide a neural progenitor spheroid, as described above. After about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days in suspension culture, the floating neural progenitor spheroids are moved to neural media to differentiate the neural progenitors. The media is supplemented with an effective dose of FGF2 and EGF. The growth factors can be provided at a concentration for each of at least about 0.5 ng/ml, at least about 1 ng/ml, at least about 5 ng/ml, at least about 10 ng/ml, at least about 20 ng/ml, up to about 500 ng/ml, up to about 250 ng/ml, up to about 100 ng/ml. [0068] To promote differentiation of neural progenitors into hCS, comprising glutamatergic neurons, after about 1 week, about 2 weeks, about 3 weeks, about 4 weeks after FGF2/EGF exposure the neural medium is changed to replace the FGF2 and EGF with an effective dose of BDNF and NT3. The growth factors can be provided at a concentration for each of at least about 0.5 ng/ml, at least about 1 ng/ml, at least about 5 ng/ml, at least about 10 ng/ml, at least about 20 ng/ml, up to about 500 ng/ml, up to about 250 ng/ml, up to about 100 ng/ml. The cortical spheroids comprise functional glutamatergic neurons. [0069] Assembloids. The hRNS can be functionally integrated with separately cultured human cortical spheroids (hCS), to form cortico-raphe nuclei assembloids (hCS-hRNS) which include glutamatergic and serotoninergic neurons. The resulting hCS-hRNS contains neural circuits between the cortex and raphe nuclei and provides for functional integration of these circuits. For example, the functionally integrated cells interact in a physiologically relevant manner, e.g. forming synapses, transmitting signals, forming multicellular structures, and the like. [0070] The cortical spheroids are co-cultured with the human raphe nuclei spheroids in neural medium under conditions permissive for cell fusion. Condition permissive for cell fusion may include culturing the hRNS and hCS in close proximity, e.g. in direct contact with one another. [0071] Assembly may be performed with spheroids after around about 30 days, about 60 days, about 90 days of culture for hRNS; and after around about 30 days, about 60 days, about 90 days of culture for hCS. The hRNS and hCS spheroids may be co-cultured for a period of 2 days, 3 days, 5 days, 8 days, 10 days, 14 days, 18 days, 21 days or more. Assembly may be carried out in neural medium. The resulting cortico-raphe nuclei assembloids are demonstrated to contain functional neural circuits, where the assembloids comprise bidirectional bidirectional projections between cortical and raphe nuclei spheroids and the serotonergic neurons of the raphe nuclei spheroids were able to modulate activity of cortical neural circuits. Methods for confirming the functionality of the neurons are known in the art and include optogenetic methods and imaging of calcium activity in neurons, such as those methods described in the examples. In some embodiments, the methods may comprise confirming the functionality of the neurons in the cortico-raphe nuclei assembloid. SCREENING ASSAYS [0072] Also disclosed herein are screening assays which involve determining the effect of a candidate agent on the spheroid, e.g. hRNS, or assembloid, e.g. hCS-hRNS, or a cell derived therefrom. A candidate agent may be a small molecule or a genetic agent. The screening assays may involve contacting the candidate agent with the spheroid, assembloid or cell derived therefrom and determining effect of the candidate agent on a parameter of the spheroid, assembloid or cell, where such parameters include morphologic, genetic or functional changes. [0073] For example, a screening assay may involve determining the effect that a candidate agent (e.g. a SSRI inhibitor) has on the functionality of neural circuits within a spheroid or assembloid. As described herein, hCS-hRNS assembloids were demonstrated to comprise neurons with bidirectional projections between the hCS-hRNS and the serotonergic neurons of hRNS were able to modulate function of the cortical neural circuits, as revealed by a combination of viral labeling and calcium imaging with photo-stimulation. The screening assays may therefore involve determining whether a candidate agent is able to alter the ability of the serotonergic neurons to modulate function of the cortical neural circuits in the hCS. [0074] As also described herein, various diseases and disorders are associated with serotonergic dysfunction. Accordingly, the assays described herein may find particular utility where the spheroid or assembloid comprise at least one allele associated with a neurologic or psychiatric disorder, schizophrenia, affective disorder (e.g. MDD, bipolar disorder or an anxiety disorder) and autism spectrum disorder (ASD). Candidate agents that are able to e.g. restore the functionality of neural circuits (e.g. cortical neural circuits) in spheroids or assembloids comprising these disorder-associated alleles may have therapeutic utility in the treatment of said disorder. [0075] Furthermore, the assembloids described herein can be used to dissect cell autonomous contributions in these disorders. For example, an assembloid can be generated where one spheroid (e.g. hRNS or hCS) is derived from a patient suffering from a disorder described herein and the other spheroid is derived from an unaffected individual, i.e. a subject not suffering from the same disorder. For example, in the methods of generating assembloids from a first and second human pluripotent stem cell, either the first or second human pluripotent stem cell can comprise at least one allele associated with a neurologic or psychiatric disorder. [0076] Neural activity causes rapid changes in intracellular free calcium. Calcium imaging assays that exploit this can therefore be used to determine the functional of neuronal circuits. This may involve modifying neurons to contain genetically-encoded calcium indicator proteins, such those proteins that include the fluorophore sensor GCaMP and imaging those cells. GCaMP comprises a circularly permuted green fluorescent protein, a calcium-binding protein calmodulin (CaM) and CaM-interacting M13 peptide, where brightness of the GFP increases upon calcium binding. Further details about calcium imaging assays are described in Chen et al. (2013) Nature 499(7458): 295-300. Other calcium imaging assays include Fura-2 calcium imaging; Fluo-4 calcium imaging, and Cal-590 calcium imaging. [0077] For example, the neurons may be modified to express GCamP6f. This can be combined with methods that activate certain neurons in response to an external stimulus, for example optogenetic methods that activate neurons in response to light. For example, to test functionality between two types of neurons involved in a neural circuit, a “first” neuron can be modified to express an optogenetic actuator (e.g. ChrimsonR) and a “second” neuron modified to express a calcium indicator (e.g. GCamP6f) and imaging used to monitor calcium release. If the first neuron is functionally connected (synapses with) the second neuron, then optogenetic activation of the first neuron will affect intracellular calcium levels and a visible readout in the second neuron. [0078] As described above, serotonin can result in various different responses, which may depend on the receptor serotonin is acting on. Thus, a method of determining the ability of serotonergic neurons to modulate cortical neural circuits may comprise labelling cells of the hRNS with an optogenetic actuator (e.g. ChrimsonR) and labelling cells of the hCS with a calcium indicator, stimulating cells of the hRNS in the hRNS-hCS assembloid and determining whether there is an increase or decrease calcium activity in cells of the hCS in the assembloid. Such increase or decrease may be transient or may occur during the entire post-stimulation period. As set out in the examples herein, such a method was used to confirm serotonergic modulation of the cortical neural circuits in the hRNS-hCS assembloid. To determine the effect a candidate agent has on this serotonergic modulation, this optogenetic method can be carried out in the presence and absence of a candidate agent and the results in the two conditions compared. [0079] Methods of analysis at the single cell level are also of interest, e.g. as described above: live imaging (including confocal or light-sheet microscopy), single cell gene expression or single cell RNA sequencing, calcium imaging, immunocytochemistry, patch-clamping, flow cytometry and the like. Various parameters can be measured to determine the effect of a drug or treatment on the spheroids, assembloids or cells derived therefrom. For example, single cell RNA sequencing of the cells that make up the spheroid or assembloid can be used to characterize the identity of these cells and can be utilized in assays that aim to determine whether a candidate agent affects cell fate. [0080] Parameters are quantifiable components of cells, particularly components that can be accurately measured, desirably in a high-throughput system. A parameter can also be any cell component or cell product including cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. While most parameters will provide a quantitative readout, in some instances a semi-quantitative or qualitative result will be acceptable. Readouts may include a single determined value, or may include mean, median value or the variance, etc. Variability is expected and a range of values for each of the set of test parameters will be obtained using standard statistical methods with a common statistical method used to provide single values. [0081] Parameters of interest include detection of cytoplasmic, cell surface or secreted biomolecules, biopolymers, e.g. polypeptides, polysaccharides, polynucleotides, lipids, etc. Cell surface and secreted molecules are a preferred parameter type as these mediate cell communication and cell effector responses and can be more readily assayed. In one embodiment, parameters include specific epitopes. Epitopes are frequently identified using specific monoclonal antibodies or receptor probes. In some cases, the molecular entities comprising the epitope are from two or more substances and comprise a defined structure; examples include combinatorically determined epitopes associated with heterodimeric integrins. A parameter may be detection of a specifically modified protein or oligosaccharide. A parameter may be defined by a specific monoclonal antibody or a ligand or receptor binding determinant. [0082] Candidate agents of interest are biologically active agents that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. An important aspect of the invention is to evaluate candidate drugs, select therapeutic antibodies and protein-based therapeutics, with preferred biological response functions. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. [0083] Also included are pharmacologically active drugs, genetically active molecules, etc. Compounds of interest include chemotherapeutic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modifiers, and neuroactive agents. Exemplary of pharmaceutical agents suitable for this invention are those described in, “The Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, New York, (1996), Ninth edition, under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Cardiovascular Drugs; Vitamins, Dermatology; and Toxicology, all incorporated herein by reference. [0084] An important class of candidate agents for use with the compositions and methods described herein are selective serotonin reuptake inhibitors (SSRIs). SSRIs are a class of drug that are typically used as antidepressants in the treatment of major depressive disorder (MDD) and anxiety disorders. SSRIs typically function by increasing the extracellular level of serotonin by limiting its reabsorption. Examples of known SSRIs include Citalopram, Escitalopram, Fluoxetine, Fluvoxamine, Paroxetine, Sertraline, Dapoxetine. As well as investigating the role of these known SSRIs, the systems described here can also be used as part of a screening assay to discover new SSRIs. [0085] Test compounds include all of the classes of molecules described above, and may further comprise samples of unknown content. Of interest are complex mixtures of naturally occurring compounds derived from natural sources such as plants. While many samples will comprise compounds in solution, solid samples that can be dissolved in a suitable solvent may also be assayed. Samples of interest include environmental samples, e.g. ground water, sea water, mining waste, etc.; biological samples, e.g. lysates prepared from crops, tissue samples, etc.; manufacturing samples, e.g. time course during preparation of pharmaceuticals; as well as libraries of compounds prepared for analysis; and the like. Samples of interest include compounds being assessed for potential therapeutic value, i.e. drug candidates. [0086] The term samples also include the fluids described above to which additional components have been added, for example components that affect the ionic strength, pH, total protein concentration, etc. In addition, the samples may be treated to achieve at least partial fractionation or concentration. Biological samples may be stored if care is taken to reduce degradation of the compound, e.g. under nitrogen, frozen, or a combination thereof. The volume of sample used is sufficient to allow for measurable detection, usually from about 0.1 to 1 ml of a biological sample is sufficient. [0087] Compounds, including candidate agents, are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. [0088] As used herein, the term “genetic agent” refers to polynucleotides and analogs thereof, which agents are tested in the screening assays of the invention by addition of the genetic agent to a cell. The introduction of the genetic agent results in an alteration of the total genetic composition of the cell. Genetic agents such as DNA can result in an experimentally introduced change in the genome of a cell, generally through the integration of the sequence into a chromosome, for example using CRISPR mediated genomic engineering (see for example Shmakov et al. (2017) Nature Reviews Microbiology 15:169). Genetic changes can also be transient, where the exogenous sequence is not integrated but is maintained as an episomal agents. Genetic agents, such as antisense oligonucleotides, can also affect the expression of proteins without changing the cell’s genotype, by interfering with the transcription or translation of mRNA. The effect of a genetic agent is to increase or decrease expression of one or more gene products in the cell. [0089] Introduction of an expression vector encoding a polypeptide can be used to express the encoded product in cells lacking the sequence, or to over-express the product. Various promoters can be used that are constitutive or subject to external regulation, where in the latter situation, one can turn on or off the transcription of a gene. These coding sequences may include full- length cDNA or genomic clones, fragments derived therefrom, or chimeras that combine a naturally occurring sequence with functional or structural domains of other coding sequences. Alternatively, the introduced sequence may encode an anti-sense sequence; be an anti-sense oligonucleotide; RNAi, encode a dominant negative mutation, or dominant or constitutively active mutations of native sequences; altered regulatory sequences, etc. The expression vector may be a viral vector, e.g. adeno-associated virus, adenovirus, herpes simplex virus, retrovirus, lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus and picornavirus vectors. [0090] Antisense and RNAi oligonucleotides can be chemically synthesized by methods known in the art. Preferred oligonucleotides are chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3’-O’-5’-S-phosphorothioate, 3’-S-5’-O-phosphorothioate, 3’-CH2-5’-O- phosphonate and 3’-NH-5’-O-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity, e.g. morpholino oligonucleotide analogs. [0091] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1:10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype. [0092] Various methods can be utilized for quantifying the presence of selected parameters, in addition to the functional parameters described above. For measuring the amount of a molecule that is present, a convenient method is to label a molecule with a detectable moiety, which may be fluorescent, luminescent, radioactive, enzymatically active, etc., particularly a molecule specific for binding to the parameter with high affinity fluorescent moieties are readily available for labeling virtually any biomolecule, structure, or cell type. Immunofluorescent moieties can be directed to bind not only to specific proteins but also specific conformations, cleavage products, or site modifications like phosphorylation. Individual peptides and proteins can be engineered to fluoresce, e.g. by expressing them as green fluorescent protein chimeras inside cells (for a review see Jones et al. (1999) Trends Biotechnol.17(12):477-81). Thus, antibodies can be genetically modified to provide a fluorescent dye as part of their structure [0093] Depending upon the label chosen, parameters may be measured using other than fluorescent labels, using such immunoassay techniques as radioimmunoassay (RIA) or enzyme linked immunoabsorbance assay (ELISA), homogeneous enzyme immunoassays, and related non-enzymatic techniques. These techniques utilize specific antibodies as reporter molecules, which are particularly useful due to their high degree of specificity for attaching to a single molecular target. U.S. Pat. No. 4,568,649 describes ligand detection systems, which employ scintillation counting. These techniques are particularly useful for protein or modified protein parameters or epitopes, or carbohydrate determinants. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules. Cell based ELISA or related non-enzymatic or fluorescence-based methods enable measurement of cell surface parameters and secreted parameters. Capture ELISA and related non-enzymatic methods usually employ two specific antibodies or reporter molecules and are useful for measuring parameters in solution. Flow cytometry methods are useful for measuring cell surface and intracellular parameters, as well as shape change and granularity and for analyses of beads used as antibody- or probe-linked reagents. Readouts from such assays may be the mean fluorescence associated with individual fluorescent antibody-detected cell surface molecules or cytokines, or the average fluorescence intensity, the median fluorescence intensity, the variance in fluorescence intensity, or some relationship among these. [0094] Both single cell multiparameter and multicell multiparameter multiplex assays, where input cell types are identified and parameters are read by quantitative imaging and fluorescence and confocal microscopy are used in the art, see Confocal Microscopy Methods and Protocols (Methods in Molecular Biology Vol.122.) Paddock, Ed., Humana Press, 1998. These methods are described in U.S. Patent no.5,989,833 issued Nov.23, 1999. [0095] The results of an assay can be entered into a data processor to provide a dataset. Algorithms are used for the comparison and analysis of data obtained under different conditions. The effect of factors and agents is read out by determining changes in multiple parameters. The data will include the results from assay combinations with the agent(s), and may also include one or more of the control state, the simulated state, and the results from other assay combinations using other agents or performed under other conditions. For rapid and easy comparisons, the results may be presented visually in a graph, and can include numbers, graphs, color representations, etc. [0096] The dataset is prepared from values obtained by measuring parameters in the presence and absence of different cells, e.g. genetically modified cells, cells cultured in the presence of specific factors or agents that affect neuronal function, as well as comparing the presence of the agent of interest and at least one other state, usually the control state, which may include the state without agent or with a different agent. The parameters include functional states such as synapse formation and calcium ions in response to stimulation, whose levels vary in the presence of the factors. Desirably, the results are normalized against a standard, usually a "control value or state," to provide a normalized data set. Values obtained from test conditions can be normalized by subtracting the unstimulated control values from the test values, and dividing the corrected test value by the corrected stimulated control value. Other methods of normalization can also be used; and the logarithm or other derivative of measured values or ratio of test to stimulated or other control values may be used. Data is normalized to control data on the same cell type under control conditions, but a dataset may comprise normalized data from one, two or multiple cell types and assay conditions. [0097] The dataset can comprise values of the levels of sets of parameters obtained under different assay combinations. Compilations are developed that provide the values for a sufficient number of alternative assay combinations to allow comparison of values. [0098] A database can be compiled from sets of experiments, for example, a database can contain data obtained from a panel of assay combinations, with multiple different environmental changes, where each change can be a series of related compounds, or compounds representing different classes of molecules. [0099] Mathematical systems can be used to compare datasets, and to provide quantitative measures of similarities and differences between them. For example, the datasets can be analyzed by pattern recognition algorithms or clustering methods (e.g. hierarchical or k-means clustering, etc.) that use statistical analysis (correlation coefficients, etc.) to quantify relatedness. These methods can be modified (by weighting, employing classification strategies, etc.) to optimize the ability of a dataset to discriminate different functional effects. For example, individual parameters can be given more or less weight when analyzing the dataset, in order to enhance the discriminatory ability of the analysis. The effect of altering the weights assigned each parameter is assessed, and an iterative process is used to optimize pathway or cellular function discrimination. [00100] The comparison of a dataset obtained from a test compound, and a reference dataset(s) is accomplished by the use of suitable deduction protocols, AI systems, statistical comparisons, etc. Preferably, the dataset is compared with a database of reference data. Similarity to reference data involving known pathway stimuli or inhibitors can provide an initial indication of the cellular pathways targeted or altered by the test stimulus or agent. [00101] A reference database can be compiled. These databases may include reference data from panels that include known agents or combinations of agents that target specific pathways, as well as references from the analysis of cells treated under environmental conditions in which single or multiple environmental conditions or parameters are removed or specifically altered. Reference data may also be generated from panels containing cells with genetic constructs that selectively target or modulate specific cellular pathways. In this way, a database is developed that can reveal the contributions of individual pathways to a complex response. [00102] The effectiveness of pattern search algorithms in classification can involve the optimization of the number of parameters and assay combinations. The disclosed techniques for selection of parameters provide for computational requirements resulting in physiologically relevant outputs. Moreover, these techniques for pre-filtering data sets (or potential data sets) using cell activity and disease-relevant biological information improve the likelihood that the outputs returned from database searches will be relevant to predicting agent mechanisms and in vivo agent effects. [00103] For the development of an expert system for selection and classification of biologically active drug compounds or other interventions, the following procedures are employed. For every reference and test pattern, typically a data matrix is generated, where each point of the data matrix corresponds to a readout from a parameter, where data for each parameter may come from replicate determinations, e.g. multiple individual cells of the same type. As previously described, a data point may be quantitative, semi-quantitative, or qualitative, depending on the nature of the parameter. [00104] The readout may be a mean, average, median or the variance or other statistically or mathematically derived value associated with the measurement. The parameter readout information may be further refined by direct comparison with the corresponding reference readout. The absolute values obtained for each parameter under identical conditions will display a variability that is inherent in live biological systems and also reflects individual cellular variability as well as the variability inherent between individuals. [00105] Classification rules are constructed from sets of training data (i.e. data matrices) obtained from multiple repeated experiments. Classification rules are selected as correctly identifying repeated reference patterns and successfully distinguishing distinct reference patterns. Classification rule-learning algorithms may include decision tree methods, statistical methods, naive Bayesian algorithms, and the like. [00106] A knowledge database will be of sufficient complexity to permit novel test data to be effectively identified and classified. Several approaches for generating a sufficiently encompassing set of classification patterns, and sufficiently powerful mathematical/statistical methods for discriminating between them can accomplish this. [00107] The data from cells treated with specific drugs known to interact with particular targets or pathways provide a more detailed set of classification readouts. Data generated from cells that are genetically modified using over-expression techniques and anti-sense techniques, permit testing the influence of individual genes on the phenotype. [00108] A preferred knowledge database contains reference data from optimized panels of cells, environments and parameters. For complex environments, data reflecting small variations in the environment may also be included in the knowledge database, e.g. environments where one or more factors or cell types of interest are excluded or included or quantitatively altered in, for example, concentration or time of exposure, etc [00109] For further elaboration of general techniques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews in cell biology, tissue culture, embryology, and neurobiology. With respect to tissue culture and embryonic stem cells, the reader may wish to refer to Teratocarcinomas and embryonic stem cells: A practical approach (E. J. Robertson, ed., IRL Press Ltd.1987); Guide to Techniques in Mouse Development (P. M. Wasserman et al. eds., Academic Press 1993); Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (P. D. Rathjen et al., Reprod. Fertil. Dev.10:31, 1998). [00110] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech. [00111] Each publication cited in this specification is hereby incorporated by reference in its entirety for all purposes. [00112] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. EXPERIMENTAL [00113] Described herein is a novel approach to study functional neuromodulatory systems using human pluripotent stem cells (hPSCs) to generate three-dimensional (3D) assembloids that contain forebrain organoids coupled to organoids that model the raphe nuclei and that are capable of sending serotonergic projections. We first generated hPSC-derived raphe nuclei spheroids (hRNS), which include functional serotonergic neurons along with other resident cell types of raphe nuclei. Assembly of human cerebral cortical spheroids (hCSs), which include pyramidal glutamatergic neurons of various cortical layers, with hRNS resulted in hRNS-hCS assembloids within which bidirectional projections between cortex and raphe nuclei are present. Using a combination of single-cell RNA sequencing, viral labelling and calcium imaging with photo- stimulation, we demonstrated an in vitro, human cellular model of serotonergic modulation of cortical circuits, which could be used as a platform for understanding the assembly of these circuits, modelling of disorders such as MDD, autism spectrum disorder and schizophrenia, and developing drug screening to identify better drugs targeting this neuromodulatory pathway. Example 1 Methods [00114] Generation of human raphe nuclei spheroids (hRNS). Human pluripotent stem cells (hPSCs) were maintained on six-well plates coated with recombinant human vitronectin (VTN-N, Life Technologies, A14700) in Essential 8 medium (Life Technologies, A1517001) supplemented with penicillin and streptomycin (1:100, Gibco, 15140122). To generate hRNS, hiPSCs were incubated with Accutase (Innovate Cell Technologies, AT-104) at 37°C for 7 min and dissociated into single cells. Approximately 3 million single cells were added per AggreWell 800 (STEMCELL Technologies, 34815) well in Essential 8 medium supplemented with the ROCK inhibitor Y-27632 (10 mM, Selleckchem, S1049), centrifuged at 100g for 3 min to capture the cells in the microwells and incubated at 37 °C with 5% CO2. After 24 hours, we collected spheroids from each microwell by firmly pipetting (with a cut end of a P1000 tip) medium in the well up and down and transferring it into ultra-low-attachment plastic dishes (Corning, 3262) in Essential 6 medium (Life Technologies, A1516401) supplemented with dorsomorphin (2.5 mM, Sigma-Aldrich, P5499) and SB-431542 (10 mM, Tocris, 1614). From day 2 to 8, medium was changed every day and supplemented with dorsomorphin, SB-431542 (10 mM from day 2 to 5, 2.5 mM from day 5 to 8) and GSK-3 inhibitor CHIR 99021 (1.5 mM). [00115] On day 6, neural spheroids were transferred to neural medium containing Neurobasal A (Life Technologies, 10888), B-27 supplement without vitamin A (Life Technologies, 12587), GlutaMax (1:100, Life Technologies, 35050) and penicillin and streptomycin. From day 5 to day 15, neural medium was changed every day and was supplemented with CHIR 99021 (1.5 mM) and Smoothened agonist SAG (100 nM). From day 8 to 20, neural medium was also supplemented with fibroblast growth factor-4 (FGF4, 10ng/mL). [00116] To promote differentiation, from day 16 to 30, neural medium was changed every other day and was supplemented with BDNF (10 ng/mL), NT-3 (10 ng/mL), IGF-1 (10 ng/mL), cAMP (100 nM), L-ascorbic acid (200 mM) and Docosahexaenoic Acid (DHA, 10 mM). A schematic showing the different recipes is presented in Fig.1A. To characterize cellular diversity in hRNS, we performed single cell transcriptomics on dissociated hRNS at day 42 based of supplier’s recommendations (10x genomics, 120262). To quantify serotonin release in hRNS, 2–3 intact hRNS per timepoint per hiPS cell line were flash frozen at various timepoints and processed for high performance liquid chromatography (HPLC). For serotonin uncaging experiments, NPEC caged-serotonin (Tocris, 3991) was used at a final concentration of 50 mM. The FRAP module of the Leica SP8 confocal microscope was used to uncage glutamate using UV light (405nm). [00117] Generation of cortico-raphe nuclei assembloids (hCS-hRNS). (Fig. 1A) To generate cortico-raphe cortico-raphe nuclei assembloids (hCS-hRNS), hCS and hRNS were generated separately, and later assembled by placing them in close proximity with each other in 1.5 ml microcentrifuge tubes for 3 days in an incubator. Neural media used for assembly contained neurobasal-A, B-27 supplement without vitamin A, GlutaMax (1:100), penicillin and streptomycin (1:100). Media was carefully changed on day 2, and on the 3 rd day, assembloids were placed in 24-well ultra-low attachment plates in the neural medium described above using a cut P1000 pipette tip. After this, media was changed every 3–4 days. hCS was generated by previously described methods13,14. Assembly was performed between days 45 and 60. For some experiments hCS or hRNS were virally labeled with AAV-DJ1-hSyn1::YFP seven to ten days prior to assembly. For optogenetic photo-stimulation experiment, hRNS were labeled with AAV1- hSyn1::ChrimsonR-tdTomato virus and assembled with EF1a-GCaMP6s-expressing hCS asvdescribed above. Example 2 Generation of functional hRNS [00118] To specify spheroids (organoids) resembling raphe nuclei, hPSCs aggregated in microwells were first patterned by double SMAD inhibition towards neuroectoderm and later exposed to CHIR, the SHH agonist SAG and FGF4 (Fig. 1B). Gene and protein expression analysis by RT-qPCR and immunocytochemistry at day 15 of patterning showed upregulation of transcription factors that drive caudal midbrain/hindbrain development (NKX6-1, NKX2-2, OLIG2, GATA2, GATA3, LMX1B, FOXA2, EN1; Fig. 1C, D) and downregulation of forebrain marker FOXG1 (Fig.1C). [00119] Immunocytochemistry at day 52 showed the presence of serotonergic neurons characterized by the presence of 5-hydroxytryptamine (serotonin, 5-HT) and one of the principle enzymes in serotonin synthesis pathway tryptophan 5-hydroxylase 2 (TPH2). The core molecular phenotype of mature serotonergic neurons includes vesicular monoamine transporter 2 (VMAT2), which packages 5-HT into synaptic vesicles and serotonin reuptake transporter SERT and recycles extracellular 5-HT15. Immunocytochemistry at day 52 revealed cells positive for both SERT and VMAT2 in hRNS (Fig.1E). Next, we used HPLC to measure 5-HT release in hRNS. Across three different timepoints and lines, we consistently measured 5-HT in hRNS ranging from 20–175 ng/mL/mg protein, whereas no detectable 5-HT was present in any of the timepoints for hCS, demonstrating the specificity of hRNS patterning towards raphe nuclei (Fig. 1F). Next, surveyed the gene expression profiles for eleven 5HT metabotropic receptors (HTRs) in hCS. We observed that a combination of excitatory Go/Gs/G11-coupled (in pink, HTR2a, HTR6, HTR2c) and inhibitory Gi/Go-coupled (in green, HTR1b, 1d) were expressed in hCS at day 100 (Fig.1G). [00120] To probe the diversity of cell types in hRNS, we performed single cell transcriptomics of hRNS on day 79-82. Unsupervised clustering of hRNS cells revealed large populations of hindbrain-lineage neurons expressing classical markers for the 5HT-lineage markers (“5HT Neurons”) as well as other neuronal subtypes (“GABAergic Neurons”, “Glutamatergic Neurons”) (Fig. 2A-B). Closer inspection of the 5-HT cluster revealed caudal and rostral subpopulations (Fig.2C) Example 3 Assembly of hCS-hRNS To model the development and function of the cortico-raphe nuclei circuitry, hRNS were virally labeled using AAV-DJ1-hSYN1::mCherry between days 45 and 60 and assembled with hCS 7-8 days later, resulting in hRNS-hCS assembloids. Live imaging of intact hRNS-hCS 16 days after assembly (days after fusion; daf) showed hRNS-derived mCherry + cells extensively projecting to hCS (Fig. 3A). Immunocytochemistry additionally showed TPH2 + cells projecting into the hCS (Fig.3B). To assess the directionality of projections in hRNS-hCS, we virally labelled hRNS and hCS with AAV-DJ1-hSYN1::eYFP and AAV-DJ1-hSYN1::mCherry respectively, and subsequently assembled them. Live imaging of intact hRNS-hCS 50 days after assembly revealed bidirectional projections between hRNS and hCS. Forebrain-projecting serotonergic cells in raphe nuclei display distinct axonal morphologies with large and oval varicosities along thin axons [4]. Similar structures were observed along the axons of hRNS-derived eYFP + projections in hCS, and not in hCS-derived mCherry+ projections (Fig.3C). Example 4 Functional probing of neuromodulatory connectivity in hCS-hRNS To label the 5HT-lineage neurons in hRNS for functional studies, we used a viral reporter that drives expression of emGFP under the FEV minipromoter Ple67 (AAV-Ple67::emGFP). We characterized its specificity using immunostainings for 5-HT and TPH2 on dissociated hRNS cells infected with Ple67::emGFP (Fig.4A); this experiments showed that 80–90% of all emGFP + were 5HT + or TPH2 + indicating high specificity. Next, we used an iCRE-dependent version of this reporter (AAV-DJ-Ple67iCRE) and co-infected hRNS with AAV-EF1Į-DIO-eYFP to induce recombination and drive eYFP expression in 5-HT-lineage cells. We then assembled co-infected hRNS with hCS infected with AAV-hSYN1::mCherry. The resulting hCS-hRNS showed extensive eYFP + projections of 5-HT-lineage cells from hRNS into hCS (Fig. 4B). To functionally probe serotonergic input into the hCS in hCS-hRNS, we used the same iCRE-dependent Ple67 reporter to express ChRmine-K V 2.1, which is a soma-targeted red-shifted opsin, in 5HT-lineage cells of hRNS (AAV-Ple67iCRE and AAV-EF1Į-DIO-ChRmine-Kv2.1) and assembled them with hCS labelled with a genetically encoded calcium indicator (AAV-hSYN1-GCamP7s). Photo-stimulation of 5-HT-lineage cells performed by high-frequency optical stimulations at the 625 nm wavelength reliably evoked responses in the hCS neurons as indicated by the stimulation-locked calcium responses (Fig.4C). CITATIONS [00121] Nadim, F. & Bucher, D. Neuromodulation of Neurons and Synapses. Curr. Opin. Neurobiol.0, 48–56 (2014). Bucher, D. & Marder, E. SnapShot: Neuromodulation. Cell 155, 482- 482.e1 (2013). Vitalis, T. & Parnavelas, J. G. The Role of Serotonin in Early Cortical Development. Dev. Neurosci.25, 245–256 (2003). Hornung, J.-P. The human raphe nuclei and the serotonergic system. J. Chem. Neuroanat.26, 331–343 (2003). Jacobs, B. L. & Azmitia, E. C. Structure and function of the brain serotonin system. Physiol. Rev. 72, 165–229 (1992). Hodge, R. D. et al. Conserved cell types with divergent features between human and mouse cortex. bioRxiv 384826 (2018). doi:10.1101/384826 Sundström, E. et al. Neurochemical differentiation of human bulbospinal monoaminergic neurons during the first trimester. Dev. Brain Res.75, 1–12 (1993). Takahash et al. Distribution of serotonin-containing cell bodies in the brainstem of the human fetus determined with immunohistochemistry using antiserotonin serum. Brain Dev.8, 355–365 (1986). Adell, A. Revisiting the role of raphe and serotonin in neuropsychiatric disorders. J. Gen. Physiol. 145, 257–259 (2015). Sodhi, M. S. K. & Sanders-Bush, E. Serotonin and brain development. in International Review of Neurobiology 59, 111–174 (Academic Press, 2004). Bonnin, A. et al. A transient placental source of serotonin for the fetal forebrain. Nature 472, 347– 350 (2011). Whitaker-Azmitia, P. M. Serotonin and brain development: role in human developmental diseases. Brain Res. Bull. 56, 479–485 (2001). Paúca, A. M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12, 671–678 (2015). Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. Nature 545, 54–59 (2017). Okaty, B. W., Commons, K. G. & Dymecki, S. M. Embracing diversity in the 5-HT neuronal system. Nat. Rev. Neurosci.1 (2019). doi:10.1038/s41583-019- 0151-3. Celada, P., Puig, M. V. & Artigas, F. Serotonin modulation of cortical neurons and networks. Front. Integr. Neurosci. 7, (2013). Frank, C. Recognition and treatment of serotonin syndrome. Can. Fam. Physician 54, 988–992 (2008). Walsh, J. J. et al.5-HT release in nucleus accumbens rescues social deficits in mouse autism model. Nature 560, 589 (2018). Doan, R. N. et al. Recessive gene disruptions in autism spectrum disorder. Nat. Genet. 1(2019). doi:10.1038/s41588-019-0433-8.
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