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

Published: September 21, 2021

doi:

Kara Lopez-Lengowski², Annie Kathuria^2,3, Kaia Gerlovin², Rakesh Karmacharya^2,3,4,5,6,7

¹Center for Genomic Medicine,Massachusetts General Hospital, ²Chemical Biology Program,Broad Institute of MIT & Harvard, ³Department of Psychiatry,Harvard Medical School, ⁴Schizophrenia and Bipolar Disorder Program,McLean Hospital, ⁵Program in Neuroscience,Harvard University, ⁶Program in Chemical Biology,Harvard University, ⁷Harvard Stem Cell Institute

Summary

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.

Abstract

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.

Introduction

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-K^band D^b)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 α (TNFα), IL-1β, IL-6, IL-10 and interferon-γ (IFN-γ) 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. 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.

Protocol

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 Prepare a 1:50 dilution of LDEV-free reduced growth factor basement membrane matrix…

Representative Results

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 (<…

Discussion

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 …

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

This work was supported by a National Institute of Mental Health Biobehavioral Research Awards for Innovative New Scientists (BRAINS) Award R01MH113858 (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 & Behavior Research Foundation (to A.K.), the Phyllis & 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.

Materials

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

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