This work was supported by a National Institute of Mental Health Biobehavioral Research Awards for Innovative New Scientists (BRAINS) Award R01MH113858&#160;(to R.K.), National Institute of Mental Health Clinical Scientist Development Award K08MH086846 (to R.K.), the Doris Duke Charitable Foundation Clinical Scientist Development Award (to R.K.), the Ryan Licht Sang Bipolar Foundation (to R.K.), the Jeanne Marie Lee-Osterhaus Family Foundation and the NARSAD Young Investigator Award from the Brain &#38; Behavior Research Foundation (to A.K.), the Phyllis &#38; Jerome Lyle Rappaport Foundation (to R.K.), the Harvard Stem Cell Institute (to R.K.) and by Steve Willis and Elissa Freud (to R.K.). We would like to thank Dr. Bruce M. Cohen and Dr. Donna McPhie from Harvard Medical School and McLean Hospital for providing us with the fibroblasts used in the study.

The human iPSCs used in this study were reprogrammed from fibroblasts that had been obtained through informed consent from healthy control subjects, with approval from the institutional review board (IRB). The reprogramming and characterization of iPSCs used in this study (ML15, ML27, ML40, ML56, ML141, ML 250, ML292) were described in a prior study51.
1. Maintenance of iPSCs
<ol>
	<li>Prepare a 1:50 dilution of LDEV-free reduced growth factor basement membrane matrix...

The development of differentiation methods along different trajectories for pluripotent stem cells have opened many avenues for the investigation of brain function and disease processes53,54,55. Initial studies had focused on the development of specific neuronal cell types hypothesized to be important in specific brain disorders56,57. Recently, brain organoids have also ...

In the human brain, microglia are the primary innate immune cells1. Brain development is regulated by microglia via two routes: release of diffusible factors and phagocytosis1. Microglia-derived diffusible factors help support myelination, neurogenesis, synaptic formation, maturation, cell death, and cell survival1. Microglia also phagocytize various elements in brain synapses, axons and in both living and dead cells2,3,4,5,6,7,8. Receptors on microglia recognize tags such as calreticulin, ATP, and sialic acid and regulate cellular phagocytosis9,10. In the hippocampus, microglia maintain the homeostasis of neurogenesis through its phagocytic role11.
Synaptic phagocytosis in the dorsolateral geniculate nucleus (dLGN) of the rodent brain has been shown to be regulated by microglia1. In rodents, it has been shown that there are two periods during the development when intense microglial synaptic phagocytosis is observed. The first period occurs during initial synapse formation and the second period occurs when connections are being fine-tuned and pruned12. Other factors that are involved in synaptic pruning are inflammatory proteins and the Class I major histocompatibility complex (MHC1, H2-Kb and Db)13,14. It has been suggested that C1q (complement component 1q) on the microglia colocalizes with MHC1, which triggers synaptic pruning15. Furthermore, mouse studies show that interleukin-33 (IL-33) secreted by astrocytes regulates synapse homeostasis in the thalamus and the spinal cord through its effects on microglia, though this has yet to be investigated in humans13. Microglia secrete a variety of cytokines that help maintain neuronal health, such as tumor necrosis factor &#945; (TNF&#945;), IL-1&#946;, IL-6, IL-10 and interferon-&#947; (IFN-&#947;) and these cytokines can modulate dendritic spine and synapse formation16,17,18. There are significant gaps in our knowledge of neuron-microglia interactions during human brain development. Most of our knowledge comes from studies from rodent models, while there is a paucity of information on the temporal and mechanistic aspects of synaptic pruning in the human cortex. Microglia support neuronal survival in the neo-cortex, and other cell types contribute as well1. It is not clear how microglia contribute to this preservation and what the interplay between microglia and the other cell types are. Microglia release several cytokines that affect neuronal and synaptic development but the mechanistic basis of their effects of these cytokines in neurons are largely unknown19,20. In order to develop a more complete understanding of the function of microglia in the human brain, it is critical to explore its interactions with different cell types found in the human brain. This report describes a method to co-culture human iPSC-derived neurons and microglia generated from the same individual. Establishing this methodology will enable well-defined investigations to interrogate the nature of microglia-neuronal interactions and to develop robust in vitro cellular models to study neuroimmune dysfunction in the context of different neurodevelopmental and neuropsychiatric disorders.
The role of microglia in schizophrenia 
Synaptic pruning is a major neurodevelopmental process that takes place in the adolescent brain21,22. Multiple lines of evidence suggest that synaptic pruning during this critical period is abnormal in schizophrenia (SCZ)23,24,25,26. SCZ is a chronic, debilitating psychiatric disorder characterized by hallucinations, delusions, disordered thought processes and cognitive deficits23,24. Microglia, the resident macrophages in the brain, play a central role in synaptic pruning25,26. Postmortem and positron emission tomography (PET) studies show evidence for dysfunctional microglial activity in SCZ25,26,27,28,29,30,31,32. Postmortem SCZ brains show well-replicated but subtle differences in the brain - pyramidal neurons in the cortical layer III show decreased dendritic spine density and fewer synapses33,34,35. Synaptic pruning is a process by which superfluous excitatory synaptic connections are eliminated by microglia during adolescence, when SCZ patients usually have their first psychotic break22,36. Postmortem studies show an association between SCZ and microglial activation, with increased density of microglia in SCZ brains, as well as increased expression of proinflammatory genes27. In addition, PET studies of human brains using radioligands for microglial activation show increased levels of activated microglia in the cortex25,26,27,28.&#160;Recent genome-wide association studies (GWAS) show that the strongest genetic association for SCZ resides in the major histocompatibility complex (MHC) locus, and this association results from alleles of the complement component 4 (C4) genes that are involved in mediating postnatal synaptic pruning in rodents37. This association has provided additional support for the hypothesis that aberrant pruning by microglia may result in the decreased dendritic spine density seen in SCZ postmortem brains. Investigations of microglial involvement in synaptic pruning in SCZ have so far been limited to indirect studies with PET imaging or inferences from investigations of postmortem brains.
Generating human microglia in the laboratory 
Cultured primary mouse microglia have been frequently used in studying microglia, though there are several indications that rodent microglia may not be representative of human microglial anatomy and gene expression (Table 1)38. Several studies have also differentiated microglia directly from blood monocytes through transdifferentiation39,40,41,42. Blood monocyte-derived microglia-like cells exhibit major differences from human microglia in gene and protein expression profile pro-inflammatory responses, and they appear to be more macrophage-like in their biology43. Recent methodological advances now enable the generation of microglia from human iPSCs, which provide opportunities to study live microglia that more accurately resemble the biology of microglia found in the human brain (Table 2). These iPSC-derived microglial cells have been shown to recapitulate the phenotype, gene expression profiles, and functional properties of primary human microglia44,45,46,47,48. This paper provides a method to co-culture human iPSC-derived neurons and microglia generated from the same individual in order to develop personalized in vitro models of neuron-microglia interactions. For this in vitro co-culture model, a microglial differentiation protocol from the Fossati group was adapted (Table 3) and combined with an adapted version of a cortical neuronal generation protocol from the Livesey group (Table 4)49,50.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>Sigma-Aldrich</td>
			<td>A6964</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>b-FGF</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-4</td>
			<td>Peprotech</td>
			<td>120-05ET</td>
			<td></td>
		</tr>
		<tr>
			<td>Brainphys</td>
			<td>StemCell Technologies</td>
			<td>5790</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11C antibody</td>
			<td>Biolegend</td>
			<td>337207</td>
			<td>Dilution 1:200</td>
		</tr>
		<tr>
			<td>Costar Flat Bottom Cell Culture Plates</td>
			<td>Corning</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Ctip2 antibody</td>
			<td>Abcam</td>
			<td>ab18465</td>
			<td></td>
		</tr>
		<tr>
			<td>CUTL1 monoclonal antibody</td>
			<td>Abnova</td>
			<td>H00001523-M01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, no phenol red</td>
			<td>Gibco</td>
			<td>21041025</td>
			<td></td>
		</tr>
		<tr>
			<td>dorsomorphin</td>
			<td>Sigma-Aldrich</td>
			<td>P5499</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D6421</td>
			<td></td>
		</tr>
		<tr>
			<td>EasYFlask Cell Culture Flasks</td>
			<td>Nunc</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Cell Lifters</td>
			<td>Fisher Scientific</td>
			<td>08-100-240</td>
			<td></td>
		</tr>
		<tr>
			<td>Flt3-Ligand</td>
			<td>Peprotech</td>
			<td>300-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo4-AM</td>
			<td>Life Technologies</td>
			<td>F-14201</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex LDEV Free RGF BME 1 ML</td>
			<td>ThermoFisher Scientific</td>
			<td>A1413201</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>ThermoFisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>Peprotech</td>
			<td>300-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti Chicken- IgG H&#38;L (Alexa Fluor 488)</td>
			<td>Abcam</td>
			<td>ab150169</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>Goat Anti mouse- IgG H&#38;L (Alexa Fluor 568)</td>
			<td>Invitrogen</td>
			<td>A-11004</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>Goat Anti Rat- IgG H&#38;L (Alexa Fluor 405)</td>
			<td>Abcam</td>
			<td>ab175670</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>Goat Anti-Guinea pig IgG H&#38;L (Alexa Fluor 405)</td>
			<td>Abcam</td>
			<td>ab175678</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14170120</td>
			<td></td>
		</tr>
		<tr>
			<td>IBA1 antibody</td>
			<td>Abcam</td>
			<td>ab5076</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>IL-34</td>
			<td>Peprotech</td>
			<td>200-34</td>
			<td></td>
		</tr>
		<tr>
			<td>INF-y</td>
			<td>Peprotech</td>
			<td>300-02</td>
			<td></td>
		</tr>
		<tr>
			<td>KiCqStart SYBR Green Primers</td>
			<td>Sigma-Aldrich</td>
			<td>KSPQ12012</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Sigma-Aldrich</td>
			<td>SML0599</td>
			<td></td>
		</tr>
		<tr>
			<td>Live Cell Imaging Solution</td>
			<td>Invitrogen</td>
			<td>A14291DJ</td>
			<td></td>
		</tr>
		<tr>
			<td>MAP2 antibody</td>
			<td>Synaptic Systems</td>
			<td>188 004</td>
			<td></td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Gibco</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>NutriStem hPSC XF Medium</td>
			<td>Biological Industries</td>
			<td>01-0005</td>
			<td></td>
		</tr>
		<tr>
			<td>P2RY12 antibody</td>
			<td>Biolegend</td>
			<td>848002</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16%</td>
			<td>Fisher Scientific</td>
			<td>50-980-488</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Orthinine</td>
			<td>Sigma-Aldrich</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>SATB2 antibody</td>
			<td>Abcam</td>
			<td>ab51502</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Sigma-Aldrich</td>
			<td>S4317</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>Stemcell Technologies</td>
			<td>78062</td>
			<td></td>
		</tr>
		<tr>
			<td>SensoPlate 24-Well Glass-Bottom Plate</td>
			<td>Greiner-Bio</td>
			<td>662892</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro-34 SFM (1X)</td>
			<td>Gibco</td>
			<td>10639011</td>
			<td></td>
		</tr>
		<tr>
			<td>TMEM119 antibody</td>
			<td>Abcam</td>
			<td>ab185333</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>TPO</td>
			<td>Peprotech</td>
			<td>300-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF</td>
			<td>Peprotech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene</td>
			<td>ThermoFisher Scientific</td>
			<td>15040066</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride (ROCK inhibitor)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

co-culturing microglia and cortical neurons differentiated from human induced pluripotent stem cells

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

Protocol Validation 
The iPSC-derived microglia were generated from seven iPSC lines over three different rounds of differentiation. Control iPSC lines ML27, ML56, ML292, and ML364 and schizophrenia iPSC lines ML40, ML141, and ML250 were utilized. Characterization of these iPSC lines have been described previously51. These iPSC-derived microglia were validated using ICC and qPCR. Microglia generated from the adapted protocol exhibited typical ramified microglial morphology (<...

Read this Scientific Article Protocol about Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells at JoVE.com

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia ...

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

Abstract