cat no | ioEA1093 Early Access
ioAstrocytes
Human iPSC-derived astrocytes
ioAstrocytes are functional human iPSC-derived astrocytes, deterministically programmed using opti-ox technology that convert consistently into defined astrocytes within days. Cells demonstrate expected stellate morphology, express key astrocytic markers (SOX9, EAAT1, S100B and Vimentin), are capable of phagocytosis, cytokine secretion, and modulation of neuronal activity in co-culture.
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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.
Benchtop benefits
Co-culture ready
ioAstrocytes support functional neuronal networks within co-culture settings, enabling in-vitro modelling of complex CNS biology.
Functional
Display key phagocytic and cytokine secretion functions as well as a demonstrable influence on neuronal network activity.
Consistent
Get reproducible results from every vial with lot-to-lot consistency of highly characterised & defined human iPSC-derived cells.
Co-culture protocol
This protocol describes a method of co-culturing ioAstrocytes with ioGlutamatergic Neurons and associated disease models to facilitate research into complex neuroglial interactions.
Modulation of neuronal activity in co-culture
(A) Immunocytochemistry image of ioAstrocytes and ioGlutamatergic Neurons in co-culture; staining shows expression of pan-neuronal marker MAP2 (green), astrocyte marker S100B (purple) and DAPI nuclear staining (blue). (B and C) High-density multi electrode measurements of the neuronal activity of mono- and co-cultures of ioGlutamatergic Neurons and ioAstrocytes, showing the active area (% of well) and mean firing rate (Hz) at different time points. ioAstrocytes were directly derived from iPSC after a 10-day reprogramming protocol (D10), and then harvested to seed with Day 0 (D0) ioGlutamatergic Neurons to establish their co-cultures. D10 ioAstrocytes and D0 ioGlutamatergic Neurons were also used to generate the respective mono-cultures.
Ready within days
Schematic overview of the timeline in the user manual
ioAstrocytes 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: 1. Stabilisation for 2 days. 2. Pre-maintenance for an additional 6 days. 3. From day 8 onwards, maintenance of cells according to the protocol and recommended media for the duration of assay requirements.
opti-ox deterministically programmed ioAstrocytes rapidly acquire an astrocyte phenotype
Time-lapse video capturing the rapid acquisition of astrocyte morphology upon thawing/plating of cryopreserved cells. The video was recorded over a 15-day time course by capturing images every 3 hours.
ioAstrocytes acquire a stellate astrocyte morphology from day 8
ioAstrocytes acquire a stellate shape with branched, elongating processes that continue to intensify. Images captured on an Incucyte at day 0 and days 2, 4, 8, 15 & 22 post-thaw. 10x magnification, 400 µm scale bar.
Rapid gain of functional activity
Phagocytosis of Zymosan bioparticles by ioAstrocytes
NanoLive video showing the ability of the ioAstrocytes to phagocytose pHrodo™ Red Zymosan BioParticles™.
pHrodo Red Zymosan BioParticles were added to cultures of ioAstrocytes at 15 post-thaw. At the start of the video, the particles are located outside the cells and due to the neutral pH of the media are non-fluorescent, but when phagocytised they are exposed to the acidic environments of intracellular organelles and fluoresce bright red. The video shows an increase of red fluorescent partials accumulating within the cell over a time course of 48 hours, demonstrating that ioAstrocytes have the capability to phagocytose.
ioAstrocytes secrete cytokines in response to stimulation
MSD multiplex immunoassay measuring whether ioAstrocytes are able to secrete a range of cytokines upon treatment with various proinflammatory stimuli. ioAstrocytes were treated with 3 different proinflammatory cocktails (T) or vehicle (V) at day 15 post-thaw and after 24 hours media samples were collected to measure the concentration of cytokines in the media.
The proinflammatory cocktails induce the secretion of most of the cytokines relative to the vehicle treated cells. Overall, the ioAstrocytes display the expected responses to the three distinct cocktails, including a strong response of Interleukin 6 (IL-6), known to be involved in neuroinflammation. Note that IFNg or IL-1b are present in two of the inflammatory cocktails and, therefore, the presence of these cytokines in the media will lead to high signals upon their detection and interfere with the measurements of their secreted forms.
Highly characterised and defined
Immunocytochemistry shows protein expression of key astrocyte markers
Click on the tabs to explore the data.
ioAstrocytes express at Day 15 and Day 22 the key astrocyte markers S100B, SOX9 (A) and Vimentin (B). DAPI was used as a nuclear stain
S100B is a multifaceted protein primarily found in astrocytes playing a key role in activation, neuroprotection, calcium homeostasis and astrocyte-neuron communication. SOX9 is critical for the differentiation of astrocytes. Vimentin is a cytoskeletal protein enriched in astrocytes.
RT-qPCR shows gene expression of key astrocyte markers
RT-qPCR data showing expression of key astrocyte markers EAAT1, SOX9, S100B and Vimentin (VIM) at four different timepoints (iPSC & D8, D15, D22). ioAstrocytes show expression of key markers from as early as day 8. Pluripotency markers POU5F1 (OCT4) & NANOG) are downregulated.
The SLC1A3 gene codes for the EAAT1 protein (Excitatory Amino Acid Transporter 1). Playing crucial roles in the regulation of glutamate neurotransmission, maintaining neuronal health and protecting against excitotoxicity.
Lot-to-lot consistency for experimental reproducibility
Bulk RNA-sequencing demonstrates high lot-to-lot consistency of ioAstrocytes
Bulk RNA-sequencing analysis was performed on three different lots of manufactured product at day 1 and day 22 post revival. Principal component analysis (PCA) represents the variance in gene expression between the three different lots of ioAstrocytes. This analysis shows lots clustering very closely (<0.5% differentially expressed genes) demonstrating high consistency at each given timepoint.
This lot-to-lot consistency of ioAstrocytes will help reduce experimental variation and increase the reproducibility of data. Colours represent the parental non-induced hiPSC cell line and the three lots of ioAstrocytes; shapes represent different timepoints.
Expression levels for specific genes of interest can be requested by contacting our team at technical@bit.bio.
Industry leading seeding density
Do more with every vial
The seeding density of our human iPSC-derived astrocytes has been optimised and validated to a recommended seeding density of 30,000 cells/cm2. This means scientists can do more with every vial and expand experimental design within budget without losing out on quality. Resulting in more experimental conditions, more repeats, and more confidence in the data. One Small vial can plate a minimum of 0.7 x 24-well plate, 1 x 96-well plate, or 1.5 x 384-well plate.
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