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

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 cells^{2,3,4,5,6,7,8}. Receptors on microglia recognize tags such as calreticulin, ATP, and sialic acid and regulate cellular phagocytosis^9,10. 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^b and 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 formation^16,17,18. 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 unknown^19,20. 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 brain^21,22. 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 deficits^23,24. Microglia, the resident macrophages in the brain, play a central role in synaptic pruning^25,26. Postmortem and positron emission tomography (PET) studies show evidence for dysfunctional microglial activity in SCZ^{25,26,27,28,29,30,31,32}. 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 synapses^33,34,35. 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 break^22,36. 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 cortex^25,26,27,28. 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 transdifferentiation^39,40,41,42. 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 microglia^{44,45,46,47,48}. 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

1. Prepare a 1:50 dilution of LDEV-free reduced growth factor basement membrane matrix in DMEM/F12 without phenol red and pre-coat a 6-well plate with 1 mL of the diluted solution for at least 2 h at 37 °C prior to thawing cell stocks.
2. Thaw cryopreserved iPSC stocks in a 37 °C water bath for 2 min. Add the cells to a 15 mL centrifuge tube containing 5 mL of DMEM/F12. Spin the cells down at 300 x g for 5 min.
3. Remove the coating solution from the pre-coated LDEV-free reduced growth factor basement membrane matrix plate and add 1 mL of stem cell medium (SCM) with 10 µM Rock inhibitor (Y-27632).
4. Resuspend the cell pellet in SCM with 10 µM Y-27632 and add to the pre-coated plate for a final volume of 2 mL of SCM plus Y-27632. Maintain iPSC cell cultures in this medium for 24 h in a 37 °C incubator.
5. Replace the medium with 2 mL of fresh SCM without Y-27632 24 h after thawing.
6. After iPSCs reach 80-90% confluence, passage them onto 75 mL flasks coated with LDEV-free reduced growth factor basement membrane matrix.
   1. Passage cells by first rinsing cells with HBSS and remove after letting sit for 30 s. Add 1 mL of non-enzymatic cell dissociation reagent and incubate for 4 min at 37 °C. Prepare the plate with SCM containing 10 µM Y-27632.
   2. Aspirate non-enzymatic cell dissociation reagent and add 1 mL of SCM + 10 µM Y-27632 to each well. Gently scrape the well with a cell lifter and obtain cells with a 1000 µL pipette.
   3. Deposit cells onto LDEV-free reduced growth factor basement membrane matrix-coated 75 mm flask in a total volume of 8 mL of SCM + Y-27632.

2. Microglia differentiation

NOTE: A schematic outlining the microglia differentiation protocol is depicted in Figure 1A. Media were warmed to room temperature before use.

1. Day 0: Perform a complete medium change with SCM medium supplemented with 80 ng/mL BMP-4. Perform daily medium changes with this same medium during Days 1-3, without any washing between medium changes.
2. Day 4: Prepare Day 4-5 medium: Hematopoietic medium (HM), supplemented with 25 ng/mL FGF, 100 ng/mL SCF, and 80 ng/mL VEGF. Remove the medium and replace with Day 4-5 medium containing 5 µM Y-27632.
   NOTE: Cells begin to float at this point - about half the cells are floating, and half are adherent.
3. Day 6: Prepare Day 6-13 medium: HM supplemented with 50 ng/mL SCF, 50 ng/mL IL-3, 5 ng/mL TPO, 50 ng/mL m-CSF, and 50 ng/mL Flt3-L. Collect the supernatant, add to a 15-mL conical tube and spin down for 8 min at 300 x g. Resuspend the pellet in a flask with 8 mL of Day 6-13 medium supplemented with 5 µM Y-27632.
4. Day 10: Add 8 mL of Day 6-13 medium on top of the existing medium.
5. Day 14: Prepare Day 14+ medium: HM supplemented with 50 ng/mL m-CSF, 50 ng/mL Flt3-L, 50 ng/mL GM-CSF. Collect the supernatant, add to a 50 mL conical tube and spin down for 8 min at 300 x g. Resuspend the pellet in 8 mL of Day 14+ medium containing 5 µM Y-27632 and continue to culture in this medium.
6. Day 18: Add 8 mL of Day14+ medium.
7. Day 22: Add 8 mL of fresh Day14+ medium without removing the existing medium.
8. Day 25: Move cells to step 2.9 or continue to maintain in Day14+ medium until day 50 of differentiation.
9. After Day 25, collect the supernatant in a 50-mL conical tube and spin down for 8 min at 300 x g.
   1. Prepare adherent medium: RPMI supplemented with 1% of 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl solution, 25 ng/mL GM-SCF and 100 ng/mL IL-34.
   2. Resuspend the pellet in the adherent medium containing 5 µM Y-27632.
   3. Plate cells at a density of 50,000 cells per cm² on 24-well plates for different experiments: LDEV-free reduced growth factor basement membrane matrix-coated plates for microglial monoculture, 10 µg/mL poly-L-ornithine and 10 µg/mL laminin coated glass imaging plates for imaging experiments.
   4. Dilute poly-L-ornithine and laminin in DPBS and add 250 µL/well for a 24 well imaging plate.
      NOTE: Cells in culture are now adherent in nature (Figure 1C).
10. Maintain cells in culture for at least 14 days, with bi-weekly medium changes. After day 14 post-adherence, cells have reached maturation and may be used for experiments.

3. Cortical neuron differentiation

NOTE: A schematic outlining the cortical neuron differentiation protocol is depicted in Figure 1G.

1. Day 0:
   1. Once iPSCs are confluent on LDEV-free reduced growth factor basement membrane matrix-coated plates, switch from SCM to a 50/50 mix of N2/B27 medium supplemented with 10 µM SB431542, 1 µM dorsomorphin, 100 nM LDN193189.
      NOTE: N2 medium consists of basal medium supplemented with 1% N-2 supplement, 1% 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl solution, 1% pen/strep. B27 medium consists of DMEM/F12 supplemented with 2% B-27 supplement, 1% 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl solution, 1% pen/strep.
   2. Change the medium with the above supplements added daily for 7 days.
2. Day 7:
   1. Pre-coat plates in LDEV-free reduced growth factor basement membrane matrix for at least 2 h.
   2. Passage cells 1:1 onto pre-coated plates. Rinse cells with 1 mL/well HBSS and remove after letting it sit for 30 s. Add 1 mL/well of cell detachment medium (e.g., Accutase) and incubate for 4-5 min at 37 °C. While incubating, prepare 15 mL conical tubes with 5 mL of DMEM.
   3. Gently pipette enzymatic dissociation agent to remove cells from the plate with a P1000 pipettor. Collect enzymatic dissociation agent and cell mixture in the 15 mL conical tube containing 5 mL DMEM.
   4. Centrifuge the tubes for 5 min at 300 x g. Resuspend the pellet in 1 mL of N2/B27 medium containing 10 µM Y-27632. Continue daily feedings with N2/B27 medium without any supplements.
3. Day 12: Passage cells 1:1 using methods described in 3.2.2. Continue daily feedings with N2/B27 medium.
4. Day 15/16: Passage cells 1:2 using methods described in 3.2.2. Continue daily feedings with N2/B27 medium.
5. Day 18/19: Passage cells 1:3 using methods described in 3.2.2. Continue daily feedings with N2/B27 medium until day 25.
6. Day 25: Feed cells with N2/B27 medium supplemented with 10 µM of DAPT.
7. Day 28: Feed cells with fresh, untreated N2/B27 medium.
8. Day 30: Passage cells using methods described in 3.2 onto the microglia culture plates and maintain in BrainPhys medium supplemented with 1% B-27 supplement.

4. Microglia/neuron co-cultures

1. Plate Day 30 cortical neurons on top of microglial cultures at a density of 50,000 cells per cm². Supplement medium with laminin 1 µg/mL to improve cell adherence.
2. Maintain cultures in a mix of 50% adherent medium and 50% Brainohys medium supplemented with 1% B-27 supplement. Perform half-medium change bi-weekly until experimentation.
3. Perform experiments after neurons reach day 60.

5. Interferon-γ treatment

1. Prepare fresh medium supplemented with 100 ng/mL IFN-γ. Add the medium and let it incubate for 24 h. Remove the medium and proceed to experimentation.

6. Immunocytochemistry

1. Fix cells in the culture dish with 100 μL of 4% paraformaldehyde at room temperature for 20 min.
2. Rinse cells in 1 mL of PBS thrice for 5 min each.
3. Add 1 mL of PBST (PBS + 0.1% Triton X) for 10 min.
4. Add blocking buffer: 1 mL of PBS plus 5% goat serum for 1 h.
5. Add primary antibody diluted in 100 μL of PBS + 1% goat serum - overnight at 4 °C. Optimize all primary antibodies accordingly.
6. Rinse cells in 1 mL of PBS thrice for 5 min each.
7. Add secondary antibody, diluted in 100 μL of PBS plus 1% goat serum - for 1 h at room temperature.

7. Spine analysis

1. Obtain images on a confocal microscope at 60x magnification.
2. Use ImageJ function NeuronJ⁵² to analyze images.
3. Obtain measurements for neurite length, spine length, and spine count through NeuronJ.

8. Calcium imaging

1. Prepare fresh medium with 3 µM Fluo-4AM dye. Incubate co-cultures in this medium for 30 min at 37 °C. Then rinse the cells with PBS, add live-cell imaging solution to the cells, and proceed to the imaging.
2. Using a confocal microscope equipped for live-cell imaging, obtain time-lapse images at 40x for 2 min. Activity can be recorded at baseline, with exposure to 15 mM glutamate, or in the setting of depolarization with 5 mM potassium chloride.
3. Using ImageJ, measure fluorescence intensity over time for individual cell bodies. Using the selection tool, select individual region of interest (ROI) surrounding each cell body. Open the ROI Manager and press Add to select. Continue to add new selections to the ROI manager.
4. Use the Set Measurements tool to measure Mean Gray Area. When the number of desired cell bodies has been added to the ROI manager, select them all and then use the Multi-Measure tool. This will provide a readout of the mean gray area for each over the time course of the video file. The exported data will give mean fluorescence intensity for the region of interest for each frame.
5. Determine fluorescence intensity ratio, F/F₀, where F is the fluorescence intensity at a given time and F₀ is the initial fluorescence intensity. F/F₀ can be graphed over time to examine spontaneous activity in neurons or examined at the maximum fluorescent intensity in the setting of stimulation.

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 processes^53,54,55. Initial studies had focused on the development of specific neuronal cell types hypothesized to be important in specific brain disorders^56,57. Recently, brain organoids have also ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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