cat no | io1021
ioMicroglia™
Human iPSC-derived microglia
ioMicroglia are human microglial cells precision reprogrammed from iPSC, using opti-ox™ technology.
Within 10 days post-revival, ioMicroglia are ready for experimentation, expressing (>90%) key microglia markers, including TMEM119, P2RY12, IBA1, TREM2, CX3CR1, CD11b, CD45, and CD14.
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Benchtop benefits
Functional
ioMicroglia display key phagocytic and cytokine secretion functions with lot-to-lot consistency.
Quick
Rapidly maturing human microglia that are ready to use within 10 days post-revival.
Co-culture compatible
Suitable for co-culture studies with neurons at 1 day post-thaw.
Ready within days
opti-ox precision reprogrammed ioMicroglia rapidly form a homogenous microglia population.
Time-lapse video capturing the rapid and homogeneous microglia phenotype acquisition upon thawing of cryopreserved ioMicroglia. 10 day time course.
Highly characterised and defined
Flow cytometry analysis of ioMicroglia shows key phenotypic marker expression
Flow cytometry analysis of day 10 ioMicroglia shows key microglia marker expression of TMEM119, P2RY12, CD14, CD45 and CD11b with a purity of above 95% for CD45, CD11b and CD14, >80% for TMEM119 and CD45, and >70% for TMEM119 and P2RY12.
ioMicroglia show key microglia marker expression
Immunofluorescent staining of day 10 ioMicroglia shows homogenous expression of P2RY12, IBA1 and TREM2, and a typical ramified morphology. DAPI counterstain (blue). Image taken at 10x magnification.
ioMicroglia show ramified morphology by day 10
DAY 1
DAY 4
DAY 6
DAY 10
Rapid morphological changes in the cells upon reprogramming, with key ramified morphology first identified by day 4 and continuing through to day 10. Day 1 to 10 post-thawing; 10x magnification; scale bar; 400 µm.
Whole transcriptome analysis demonstrates high lot-to-lot consistency of ioMicroglia
Bulk RNA sequencing analysis was performed on three independent lots of ioMicroglia at three different time points throughout the reprogramming protocol. Principal component analysis represents the variance in gene expression between the lots of ioMicroglia. This analysis shows high consistency between each lot of ioMicroglia at each given timepoint. Populations of ioMicroglia with equivalent expression profiles can be generated consistently from every vial, allowing confidence in experimental reproducibility.
Whole transcriptome analysis demonstrates that ioMicroglia are highly similar to primary adult, foetal and other iPSC-derived microglia
Principal component analysis of bulk RNA sequencing data from ioMicroglia, integrated with sequencing data from Abud et al. (1) shows that ioMicroglia cluster closely to primary foetal and adult microglia data sets derived from this publication. Shapes represent the experiment from which data was obtained and colours represent the cell type.
(1) Abud E, et al., Neuron, 2018; 94(2): 278-293
ioMicroglia display key functions with consistency
Phagocytosis of E. coli particles by ioMicroglia
Day 10 ioMicroglia from three independent lots were incubated with 1 µg/0.33 cm2 pHrodo™ RED labelled E. coli particles for 24 hours +/- cytochalasin D control. Images were acquired every 30 mins on the Incucyte® looking at red fluorescence and phase contrast. The graph displays the proportion of cells phagocytosing E. coli particles over 24 hours. Three technical replicates were performed per lot.
Degree of phagocytosis of E. coli particles by ioMicroglia
Day 10 ioMicroglia from three independent lots were incubated with 1 µg/0.33 cm2 pHrodo™ RED labelled E. coli particles for 24 hours +/- cytochalasin D control. Images were acquired every 30 mins on the Incucyte® looking at red fluorescence and phase contrast. The graph displays the fluorescence intensity per cell displaying degree of phagocytosis per cell, data from three independent lots. Three technical replicates were performed per lot.
Phagocytosis of Amyloid Beta-42 particles by ioMicroglia
Day 10 ioMicroglia were incubated with 500 nM AF488 labelled Aβ42 +/- cytochalasin D for 20 hours with images acquired every 30 mins on the Incucyte® and degree of phagocytosis calculated based on fluorescence. Three technical replicates were performed.
Day 10 ioMicroglia were stimulated with LPS 100 ng/ml and IFNɣ 20 ng/ml for 24 hours or pHrodo™ RED labelled E. coli particles. Supernatants were harvested and analysed using MSD V-plex Proinflammatory Kit™. ioMicroglia secrete TNF⍺, IL-6, IL-8, IL-1b, IL-12p70 and IL-10 in response to stimuli. Predominantly producing a pro-inflammatory response. This is consistent across two independent lots. Three technical replicates were performed per lot.
ioMicroglia show a pro-inflammatory cytokine response to LPS and dexamethasone treatment
Day 10 ioMicroglia were stimulated with increasing concentrations of LPS (10, 30, 100 and 300 ng/ml) +/- dexamethasone. Supernatants were harvested at 6 or 24 hours post-stimulation and analysed using the MSD V-plex Proinflammatory Kit™. ioMicroglia secrete TNF-⍺, IL-6, IL-10, IL-8, IL-1β and IL-12p70 upon treatment with LPS and inhibition is observed when treated with dexamethasone except for IL-10, as expected. ioMicroglia predominantly produce a pro-inflammatory response. Bars represent an average of n=3 replicates with standard deviation. This data was generated by Malika Bsibsi, Kimberly Lo, Matteo Zanella, Lieke Geerts, and Stefan Kostense from Charles River Laboratories (2).
(2) Malika Bsibsi, Kimberly Lo, Matteo Zanella, Lieke Geerts, and Stefan Kostense, Charles River Laboratories, Darwinweg 24, 2333 Leiden, The Netherlands.
(2) Malika Bsibsi, Kimberly Lo, Matteo Zanella, Lieke Geerts, and Stefan Kostense, Charles River Laboratories, Darwinweg 24, 2333 Leiden, The Netherlands.
ioMicroglia show a pro-inflammatory cytokine response to LPS and Amyloid Beta stimulation
Day 14 ioMicroglia were stimulated with either 0.5 µM of β-amyloid or 100 ng/ml of LPS. Supernatants were harvested after 24 hours and analysed with a custom Human Luminex® Discovery Assay kit (R&D Systems). ioMicroglia secrete various pro-inflammatory cytokines and chemokines including TNF-⍺, IL-1β, IL-6, IL-10, CCL2, CCL3, CCL5, and CXCL1 following stimulation. Bars represent an average of n=2 or n=3 replicates with standard error. This data was generated by Eve Corrie and Emma V. Jones from Medicines Discovery Catapult (3).
(3) Eve Corrie, Emma V. Jones, Medicines Discovery Catapult, Block 35, Mereside, Alderley Park, Macclesfield, SK10 4ZF. UK.
(3) Eve Corrie, Emma V. Jones, Medicines Discovery Catapult, Block 35, Mereside, Alderley Park, Macclesfield, SK10 4ZF. UK.
ioMicroglia are co-culture compatible
ioGlutamatergic Neurons were cultured to day 10 post-thaw. ioMicroglia cultured to either day 1 or day 10 post-thaw were added directly to day 10 ioGlutamatergic Neurons. The co-cultures were maintained for a further 8 days. Imaging was performed in 30-minute intervals. Representative video showing that ioMicroglia form a stable co-culture with ioGlutamatergic Neurons.
Key marker expression in ioMicroglia and ioGlutamatergic Neuron co-cultures
MAP2
IBA1
MERGE
Immunofluorescent analysis at day 8 of the co-cultures shows expression of the microglia marker, IBA1 (yellow) and the pan-neuronal marker, MAP2 (red), as expected. Representative images taken at 10x magnification with 100 μm scale bar.
Representative video showing ioMicroglia in co-culture with ioGlutamatergic Neurons selectively phagocytosing pHrodo™ Red labelled Zymosan particles, without any adverse effects on neuron morphology. ioMicroglia start to fluoresce red when they have engulfed material which is initiated by a shift in pH.
Cells arrive ready to plate
ioMicroglia are delivered in a cryopreserved format and are programmed to rapidly mature upon revival in the recommended media. The protocol for the generation of these cells is a four-phase process: Phase 0: an Induction phase that is carried out at bit.bio, Phase 1: Stabilisation for 24 hours, Phase 2: Maturation for a further 9 days, Phase 3: the Maintenance phase. Cells are ready to use from day 10.
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Beyond neurons - microglia cells and their role in neurodegeneration and neurodevelopment
An interview with a leading researcher and microglia expert Dr Anthony Vernon at King's College Institute of Psychiatry, Psychology & Neuroscience, to demystify the complex roles of microglia in our brand new blog.
