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cat no | io1021

ioMicroglia^™

Male human iPSC donor-derived microglia

ioMicroglia (io1021) are male donor-derived 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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Confidently investigate your phenotype of interest across multiple clones with our disease model clone panel. Detailed characterisation data (below) and bulk RNA sequencing data (upon request) help you select specific clones if required.

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Benchtop benefits

Functional

ioMicroglia display key phagocytic and cytokine secretion functions with lot-to-lot consistency.

Quick

Rapidly maturing male donor-derived 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 from a male donor rapidly form a homogenous microglia population.

Time-lapse video capturing the rapid and homogeneous microglia phenotype acquisition upon thawing of cryopreserved male donor-derived ioMicroglia. 10 day time course.

Highly characterised and defined

Flow cytometry analysis of male donor-derived ioMicroglia shows key phenotypic marker expression

ioMicroglia show key phenotypic marker expression

Flow cytometry analysis of day 10 male donor-derived 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.

The cell detachment protocol used to generate this data is available here.

Male donor-derived ioMicroglia show key microglia marker expression

Immunofluorescent staining of day 10 male donor-derived ioMicroglia shows homogenous expression of P2RY12, IBA1 and TREM2, and a typical ramified morphology. DAPI counterstain (blue). Image taken at 10x magnification.

Male donor-derived 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; 100x magnification; scale bar; 400 µm.

Whole transcriptome analysis demonstrates high lot-to-lot consistency of male donor-derived ioMicroglia

Bulk RNA sequencing analysis was performed on three independent lots of male donor-derived ioMicroglia at three different time points throughout the reprogramming protocol. Principal component analysis represents the variance in gene expression between the lots and shows the high consistency across each lot at each given time point. Populations of male donor-derived ioMicroglia with equivalent expression profiles can be generated consistently from every vial, allowing confidence in experimental reproducibility.

Whole transcriptome analysis demonstrates that male donor-derived ioMicroglia are highly similar to primary adult, foetal and other iPSC-derived microglia

Principal component analysis of bulk RNA sequencing data from male donor-derived ioMicroglia, integrated with sequencing data from Abud et al. (1) shows that these cells 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

Key functions with consistency

Phagocytosis of E. coli particles by male donor-derived ioMicroglia

Day 10 male donor-derived 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 male donor-derived ioMicroglia

Day 10 male donor-derived 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 male donor-derived ioMicroglia

Day 10 male donor-derived 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.

Male donor-derived ioMicroglia secrete pro-inflammatory cytokines upon activation

Day 10 male donor-derived 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™. These cells 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.

The stimulation for cytokine release protocol used to generate this data is available here.

Male donor-derived ioMicroglia show a pro-inflammatory cytokine response to LPS and dexamethasone treatment

Day 10 male donor-derived 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™. These cells 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. These cells 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.

Male donor-derived ioMicroglia show a pro-inflammatory cytokine response to LPS and Amyloid Beta stimulation

Day 14 male donor-derived 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). These cells 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.

Co-culture compatible

Male donor-derived ioMicroglia form co-cultures with ioGlutamatergic Neurons

ioGlutamatergic Neurons (io1001) were cultured to day 10 post-thaw. Male donor-derived ioMicroglia (io1021) 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 these cells form a stable co-culture with ioGlutamatergic Neurons.

The co-culture protocol used to generate this data is available here.

Key marker expression in male donor-derived 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.

The co-culture protocol used to generate this data is available here.

Male donor-derived ioMicroglia retain phagocytic function in co-culture with ioGlutamatergic Neurons

Representative video showing male donor-derived 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.

The co-culture protocol used to generate this data is available here.

Cells arrive ready to plate

bit.bio_ioMicroglia_timeline_horizontal_withoutdox

Male donor-derived 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 three-phase process: 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.

Product information

Starting material

Human iPSC line

Seeding compatibility

6, 12, 24, 96 & 384 well plates

Shipping info

Dry ice

Donor

Caucasian adult male (skin fibroblast)

Vial size

Small: >1.5 x 10⁶ viable cells
Large: >5 x 10⁶ viable cells

Quality control

Sterility, protein expression (ICC), functional phagocytosis and cytokine secretion assays

Differentiation method

opti-ox cellular reprogramming

Recommended seeding density

37,000 to 39,500 cells/cm²

User storage

LN2 or -150°C

Format

Cryopreserved cells

Product use

ioCells are for research use only

Applications

Neurodegenerative disease modeling
Drug discovery and development
Neuroinflammation modelling
Phagocytosis assays
Cytokine response assays
Co-culture studies
Transcriptome analysis