cat no | io1029

ioMicroglia^™

Female human iPSC donor-derived microglia

ioMicroglia (io1029) are female 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 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 female 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 female donor rapidly form a homogenous microglia population.

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

Highly characterised and defined

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

Flow cytometry analysis of day 10 female donor-derived ioMicroglia shows key microglia marker expression of CD11b, CD45, CD14, and P2RY12 with a purity of above 97% for all these markers.

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

Female donor-derived ioMicroglia show key microglia marker expression

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

Female donor-derived ioMicroglia show ramified morphology by day 10

ioMicroglia female morphology panel FINAL 4

Rapid morphological changes in the female donor-derived 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.

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

ioMicroglia Female bulk RNA seq lot to lot consistency PCA plot_FINAL3

Bulk RNA sequencing analysis was performed on three independent lots of female 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 female donor-derived ioMicroglia with equivalent expression profiles can be generated consistently from every vial, allowing confidence in experimental reproducibility.

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

ioMicroglia Female bulk RNA seq benchmarking PCA plot_FINAL3

Principal component analysis of bulk RNA sequencing data from female 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

Female donor-derived ioMicroglia display a different level of phagocytosis than male donor-derived cells

ioMicroglia Female Zymosan phagocytosis proportion graph_FINAL

Day 10 female donor-derived ioMicroglia (io1029) from three independent lots and male donor-derived ioMicroglia (io1021) from one lot were incubated with pHrodo™ RED labelled Zymosan particles for 24 hours +/- cytochalasin D control. The graph displays that the proportion of cells phagocytosing Zymosan particles over 24 hours is consistent across three independent lots and that female donor-derived ioMicroglia cells display a higher proportion of phagocytosis than male donor-derived cells. Images were acquired every 30 mins on the Incucyte^® looking at red fluorescence and phase contrast. Three technical replicates were performed per lot.

Female donor-derived ioMicroglia display a consistent degree of phagocytosis across lots

ioMicroglia Female Zymosan phagocytosis intensity graph_FINAL

Day 10 female donor-derived ioMicroglia (io1029) from three independent lots and male donor-derived ioMicroglia (io1021) from one lot were incubated with pHrodo™ RED labelled Zymosan particles for 24 hours +/- cytochalasin D control. The graph displays that the degree of cells phagocytosing Zymosan particles over 24 hours is consistent across three independent lots. Images were acquired every 30 mins on the Incucyte^® looking at red fluorescence and phase contrast. Three technical replicates were performed per lot.

Female donor-derived ioMicroglia phagocytose Zymosan particles

Representative video showing female donor-derived ioMicroglia (io1029) phagocytosing pHrodo™ Red labelled Zymosan particles. When female donor-derived ioMicroglia engulf these particles this causes the particles to fluoresce red, within the cells, due to the drop in pH in the phagolysosome. Live imaging was performed in 2-minute intervals over a time period of 2 hours using the 3D Cell Explorer 96focus Nanolive Imaging system.

Female donor-derived ioMicroglia display a different pro-inflammatory cytokine response to male donor-derived cells

ioMicroglia Female_cytokine secretion graphs_FINAL 2

Day 10 female donor-derived ioMicroglia (io1029) from three independent lots and male donor-derived ioMicroglia (io1021) from one lot 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™. Female donor-derived ioMicroglia (io1029) 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 three independent lots. Female donor-derived ioMicroglia (io1029) show a higher level of secretion of IL-8 and IL-1β cytokines, and a lower level of IL-12p70 cytokine than male donor-derived ioMicroglia (io1021). Three technical replicates were performed per lot.

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

Co-culture compatible

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

ioGlutamatergic Neurons (io1001) were cultured to day 10 post-thaw. Female donor-derived ioMicroglia (io1029) 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 6 days. Representative video showing that female donor-derived ioMicroglia form a stable co-culture with ioGlutamatergic Neurons. Live imaging was performed in 6.5-minute intervals over a time period of 3 hours and 31 minutes using the 3D Cell Explorer 96focus Nanolive Imaging system.

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

Key marker expression in female donor-derived ioMicroglia and ioGlutamatergic Neuron co-cultures

ioMicroglia female coculture ICC panel FINAL

Immunofluorescent analysis at day 8 of the co-cultures shows expression of the microglia marker, IBA1 (green) and the pan-neuronal marker, MAP2 (red), as expected. Representative images taken at 10x magnification.

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

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

Representative video showing female-derived donor ioMicroglia (io1029) in co-culture with ioGlutamatergic Neurons (io1001) selectively phagocytosing pHrodo™ Red labelled Zymosan particles after 10 days in co-culture, without any observed adverse effects on neuron morphology. When female donor-derived ioMicroglia engulf these particles this causes the particles to fluoresce red, within the cells, due to the drop in pH in the phagolysosome. Live imaging was performed in 8-minute intervals over a time period of 1 hour and 36 minutes using the 3D Cell Explorer 96focus Nanolive Imaging system.

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

Cells arrive ready to plate

bit.bio_ioMicroglia_timeline_horizontal_withoutdox

Female 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

Asian-Indian female (blood endothelial progenitor cells - EPCs)

Vial size

Small: >1.5 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 modelling
Drug discovery and development
Neuroinflammation modelling
Phagocytosis assays
Cytokine response assays
Co-culture studies
Transcriptome analysis