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cat no | ioA021 Early Access

ioMicroglia^™

Human iPSC-derived microglia

ioMicroglia are human induced pluripotent stem cell (iPSC)-derived microglia, precision reprogrammed using opti-ox™ technology. Within 10 days post-revival, ioMicroglia are ready for experimentation, expressing (>90%) key phenotypic markers, including TMEM119, P2RY12, IBA1, CX3CR1, CD11b, CD45, and CD14. ioMicroglia display key functional traits including phagocytosis and proinflammatory cytokine secretion, and can be co-cultured with ioGlutamatergic Neurons™ with retention of phagocytic function.

ioMicroglia provide a rapid, consistent, functional, and easy-to-use hiPSC-based model for neurodegenerative disease research and drug development.

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Benchtop benefits

Quick

Rapidly maturing human microglia that are ready to use within 10 days post-revival.

Functional

ioMicroglia display key phagocytic and cytokine secretion functions.

Co-culture compatible

Suitable for co-culture studies with neurons.

Product information

Starting material

Human iPSC line

Seeding compatibility

6, 12, 24 & 96 well plates

Shipping info

Dry ice

Donor

Caucasian adult male (skin fibroblast)

Vial size

Small: >1.5 x 10⁶ viable cells

Quality control

Sterility, protein expression (ICC), 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

Disease modeling
Drug development
Neuroinflammation research
Co-culture studies
Transcriptome analysis

Ready within days

ioMicroglia generated by transcription factor-driven reprogramming of iPSCs using opti-ox technology

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

ioMicroglia show 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.

ICC analysis of ioMicroglia shows key phenotypic marker expression

P2RY12

IBA1

MERGE

Immunocytochemistry staining of day 10 ioMicroglia shows homogenous expression of P2RY12 and IBA1, 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; 400µm.

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

ioMicroglia are consistent, and similar to gold standard microglia

Principal component analysis (PCA) of bulk RNA sequencing data from ioMicroglia, integrated with sequencing data from Abud et al. (1) and Chen et al. (2), show that ioMicroglia cluster closely to primary foetal and adult microglia data sets derived from both these publications. 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

(2) Chen SW, et al., Stem Cell Reports, 2021; 16(5): 1363-1380

ioMicroglia display key functions with consistency

Phagocytosis of E. coli particles by ioMicroglia

Proportion of cells undergoing phagocytosis over time

Day 10 ioMicroglia 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. Graph displays the proportion of cells phagocytosing E. coli particles over 24 hours. Three technical replicates were performed per batch.

Degree of phagocytosis of E. coli particles by ioMicroglia

Day 10 ioMicroglia 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 two independent batches. Three technical replicates were performed per batch.

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 per batch.

ioMicroglia secrete proinflammatory cytokines upon activation

Day 10 ioMicroglia were stimulated with LPS 100ng/ml and IFNɣ 20ng/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 proinflammatory response. This is consistent between two independent batches. Three technical replicates were performed per batch.

ioMicroglia are co-culture compatible

ioMicroglia form co-cultures with ioGlutamatergic Neurons

Video showing cryopreserved ioMicroglia and ioGlutamatergic Neurons that were thawed and cultured independently for 10 days before being co-cultured for 8 days. Imaging was then performed in 30-minute intervals. ioMicroglia form a stable co-culture with ioGlutamatergic Neurons.

Key marker expression in ioMicroglia and ioGlutamatergic Neuron co-cultures

MAP2

IBA1

MERGE

Immunocytochemistry at day 18 shows expression of MAP2 (ioGlutamatergic Neurons) and IBA1 (ioMicroglia), within the co-culture. Images taken at 10x magnification.

ioMicroglia retain phagocytic function in co-culture with ioGlutamatergic Neurons

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

bit.bio-iomicroglia_horizontal_v4-rgb-retina

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.

Supporting documentation

Statement of Use

Product resources

Webinar

Rethinking Developmental Biology With Cellular Reprogramming

Mark Kotter | CEO and founder | bit.bio

Marius Wernig | Professor Departments of Pathology and Chemical and Systems Biology | Stanford University

2023

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Poster

Genotype and phenotype validation of an isogenic human iPSC-derived neuronal model of Huntington’s Disease

V1
bit.bio

2022

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Webinar

Alzheimer’s Disease Pathogenesis: Emerging Role of Microglia

University of Münster & bit.bio

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Infographic

Comparing Stem Cells Differentiation Methods | Infographic

bit.bio

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