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

Published: September 21st, 2021



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

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 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.

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,

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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.......

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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 (<.......

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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 .......

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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....

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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

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