Human ipsc-derived Microglia

Transcription

1 Human ipsc-derived Microglia Morphological Characterization Immunocytochemistry Comparison with Macrophages Phagocytosis and Efferocytosis ax1666 HiPSC-derived Microglial Precursors ax0666 Plated HiPSC-derived Microglia ax0660 HiPSC-derived Microglia Maintenance Medium Kit

2 Microglia are commonly described as the immune cells of the brain. Under physiological conditions, they are vigilant guard keepers of their micro environment, seeking out invading pathogens and clearing up cell debris, apoptotic cells, and misfolded proteins by migrating, phagocytosing, and producing cytokines and neurotrophins; all to maintain a homeostatic balance in the CNS. Human ipsc-derived microglial precursors (monocytes) were seeded using microglia maintenance medium into 96-well plates at a density of 100,000 cells/cm2. During maturation, spherical monocytes adhere strongly to the culture surface, displaying increasingly ramified morphology as they differentiate to microglia. Axol s ipscderived microglia show dynamic surveillance behaviour, which is representative of their phenotype in vivo. An increasing body of evidence 1,2,3 linking inflammation and neurodegeneration has continued to push microglia to the forefront of neuroscience research in recent years. To date, much of microglia research has been conducted using animal models, immortalized microglia-like cell lines or primary human microglia. Each of these models has many caveats ranging from limited cell number and availability, variability in homogeneity and unrepresentative inflammatory responses. Consequently, there is a pressing need for more practical, physiologically-relevant models to study microglial behaviour. Axol s human ipsc-derived microglia provide a reproducible, physiologically-relevant model in an easy-to-use, assay-ready format for investigating neuroglia involvement in neurodegeneration and neurodevelopment. We also offer a fully optimized serum-free microglia maintenance medium to promote and support the maintenance of microglia. Axol s method for generating ipsc-derived microglia mimics the in vivo pathway of development for brain resident macrophages and produces microglia that are representative of primary human microglia in vitro. Our ipsc-derived microglia deliver the advantage that they provide an almost infinite source of microglia from a single donor with a normal karyotype. Immunocytochemistry Human ipsc-derived microglia show protein expression of myeloid and microglia-specific markers IBA-1, CX3CR1, P2RY12 and TMEM119. Recent advances in stem cell technologies have the potential to generate patient-specific microglia from human induced pluripotent stem cells (hipscs). Modelling the interface between the nervous and immune system can enable the development of novel drug discovery targets in the fields of neurodegeneration and neuroinflammation. These hipscderived microglia can therefore offer several advantages over traditional immortalised cell lines (e.g. BV-2) and primary sources, and an enhanced physiologically-relevant cellular model to advance disease research, facilitating the discovery of treatments modulating inflammatory pathways. Morphological characterization Brightfield images of Axol s human ipsc-derived microglia show typical morphology with long ramified processes characteristic of human microglia

3 Immunocytochemistry of human ipsc-derived microglia, after 18 days in culture, revealed the expression of IBA-1 and CX3CR1 (a chemokine receptor) as well as the microglial-specific markers TMEM119 (a transmembrane protein), and P2RY12 (purinergic receptor). All 3 markers co-localized strongly with the myeloid marker IBA-1, confirming identity of brain-residentlike microglia. Axol s ipsc-derived microglia are a homogenous population. (184/194) x100 =~95% Pure Population. Differentiation yields microglia that are >95% pure population as assessed by proportion of cells expressing IBA-1 versus P2RY12, in line with similar findings from other groups (Abud et al., 2017; DOI /j. neuron ). Microglia and macrophages from the same donor were stained in parallel for the macrophage/microglia markers IBA-1 (voltage-gated Ca2+ sensor) and CX3CR1 (chemokine receptor) as well as microglial-specific markers P2RY12 (purinergic receptor) and TMEM119 (transmembrane protein). Expression of P2RY12 and TMEM119 is either absent or significantly less in macrophages compared to microglia, confirming microglial identity. x10 Magnification; Scale bar=400um. Microglia comparison with macrophages Axol s ipsc-derived microglia express expected myeloid markers and are enriched for microglial-specific genes. q-rt-pcr was used to determine the mrna expression of 6 key microglia specific genes in ipscs, ipsc-monocytes, ipsc-macrophages and ipsc-microglia. Phase contrast image (left) and IBA-1-stained microglia (right) after 18 days in culture highlights typical ramified morphology of resting microglia (x20 Magnification, Scale bar=400μm). Cells were lysed from starting ipscs, day 0 (monocytes), day 14 (macrophages) or day 18 (microglia) prior to RNA isolation followed by RT. CQ1A, MERTK, P2RY12, GPR34, and TMEM119 are all differentially up-regulated in microglia. The disease-associated marker TREM2 is expressed at roughly constant levels across the myeloid cell lineage. Flow cytometry analysis confirms expression of IBA-1 and the myeloid marker CD68 (FACs was carried out using a BD Canto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar). Blue trace represents stained cells and the red trace is the unstained control. Fold change was calculated using the ΔΔCT method, with PDGB as an endogenous control and normalised to ipsc. N=3 (ipsc), N=2 (macrophage), N=1 (monocyte,microglia), technical replicates were performed in triplicate (monocyte, macrophage, microglia) or duplicate (ipsc). Error bars are +- S.E.M. FACs analysis kindly provided by Grünenthal GmbH, Germany

4 Axol s Human ipsc-derived microglia secrete cytokines in response to inflammatory stimuli. Secretion of the pro-inflammatory cytokine, Tumour Necrosis Factor alpha (TNF-α), was measured from the cellular supernatants of Axol s human ipsc-derived microglia and human ipsc-derived macrophages, using an AlphaLISA. Both microglia and macrophages, from the same donor, showed secretion of TNF-α exhibiting physiological function. Data kindly provided by Sygnature Discovery. phrodo is a fluorogenic dye that dramatically increases in fluorescence as the ph of the surroundings becomes more acidic, such is the case in the lysosome. phrodo Green Zymosan BioParticles (Life Technologies) were added at 0.5 mg/ml to day 19-microglia plated at 1x105sup>/cm2, and incubated for 3hrs at 37ºC, 5% CO2. Nuclei were counterstained with a live Hoechst stain and live cell imaging was carried out using an on-stage incubator connected to an EVOS Fl Auto imaging system (Life Technologies). Two activated microglial cells can be seen consuming increasing numbers of BioParticles over time, leading to greater and more intense fluorescence emission. Microglia seeded at 1x105/cm2 (A) or 3x105cm2 (B) were pre-treated with 100 ng/ml lipopolysaccharide (LPS) for 1hr (B), 5, 10μM or 30μM Cytochalasin D (CytoD, phagocytosis inhibitor) or 1μM or 10μM Latrunculin A (Lat-A) for 30mins (A) or 1hr (B) before adding phrodo Green Zymosan BioParticles at 0.5 mg/ml and incubating for 3hrs at 37ºC, 5% CO2. A B Lat-A inhibited phagocytosis more strongly than CytoD, in line with Lat-A being a more potent inhibitor of actin polymerization. Fluorescence was quantified using a SpectraMax id3 microplate reader (Molecular Devices), and values were blanked against a no-cell control well. N=4 for both experiments, One-way ANOVA *P<0.05, **P<0.01, ***P< Microglia engulfment of apoptotic Neuro2A Phagocytosis and Efferocytosis Phagocytosis was used to measure functional activation and inhibition of human ipsc-derived microglia. Phagocytic activity was assessed using phrodo green Zymosan bioparticles and human ipsc-derived microglia were found to be highly phagocytic. Representative image series taken over two hours of ipsc-derived microglia (Axol BioScience) engulfing IncuCyte phrodo Orange labelled apoptotic Neuro2A cells. Images verify the entry of an apoptotic target cell into the cytoplasm of the microglia. Neuro2A target cells were labelled with the IncuCyte phrodo Orange cell labelling kit and apoptosis induced with staurosporine

5 Efferocytosis of phrodo labeled apoptotic Neuro2A cells and phagocytosis of phrodo labeled E. coli bioparticles Phagocytosis of aggregated neurodegenerative disease-associated peptides 30K microglia/well were treated with increasing concentrations of either IncuCyte phrodo red E. coli bioparticles ( µg/well), or phrodo Orange labeled apoptotic Neuro-2A cells (6,250 50,000 cells/well) to measure phagocytic and efferocytic activity, respectively. 30K microglia/well were exposed to a concentration range of labelled, aggregated peptides β Amyloid ( µg/ml), α-synuclein ( µg/ml), Myelin basic protein ( µg/ml). Phase and fluorescent images were acquired over 24h and increasing fluorescence intensity was observed inside the cells. Differential rates of uptake were observed for the peptide aggregates. Images at 1, 1.5, and 24 hours show fluorescent image mask with the phase channel turned off. As target cells become engulfed by microglia, fluorescence increases as indicated by the fluorescence mask at increasing time-points. Microglia display a density dependent increase in phagocytic activity. Kinetic graphs (left) show microglia temporal response to target cells at increasing concentrations. 24-hour end point graphs (right) display the concentration dependent response. IncuCyte, Essen BioScience and all names of Essen BioScience products are registered tradesmarks and the property of Essen BioScience unless otherwise specified. Essen BioScience is a Sartorius Company. phrodo and BioParticles are registered trademarks of Life Technologies Corporation, a Thermo Fisher Scientific Company. Data kindly provided by Essen Bioscience

6 Cytokine response Images taken during phagocytosis assay showing the effect of LPS-induced clustering can be seen with several microglia in close proximity responding to and engulfing the phrodo Green Zymosan BioParticles. ipsc-microglia release cytokines IL-6 and TNFα upon stimulation. Microglia plated at 120,000/cm2 were stimulated with increasing concentrations of LPS, R848, IL1β, and IFNγ for 20hrs. The concentration of TNFα (A) and IL-6 (B) in the harvested supernatant were measured by alphalisa. Except for LPS, there is a clear dose-dependent, positive correlation between simulant concentration and amount of cytokine released. N=1. Data kindly provided by Grünenthal GmbH, Germany. A Phagocytosis analysis revealed activation of microglia after addition of phrodo green zymosan particles (0.15 mg/ml) and inhibition after treatment with CytoD (10 μm), however, no further stimulation was observed when stimulated with LPS (100 ng/ml). B Stimulation with TLR agonists LPS (TLR1/2) and R848 (TLR7/8), as well as the cytokine IL1β and interferon gamma (IFNγ) induces changes in TNFα and IL-6 release. Co-culture of ipsc-microglia with ipsc-cortical neurons Immunocytochemistry of human ipsc-derived microglia cocultured with human ipsc-derived cortical neurons reveals microglia specific maker expression TMEM119 and neuronal marker β-3 Tubulin. Relative fluorescence was significantly decreased after treatment with CytoD, statistical analysis one-way ANOVA *P<0.05. phrodo and BioParticles are registered trademarks of Life Technologies Corporation, a Thermo Fisher Scientific Company. Cytokine response was analysed from human ipscderived microglia co-cultured with human ipsc-derived cortical neurons from the same donor, after stimuli with increasing concentrations of LPS. The co-culture were incubated with LPS for 5 hours at 37ºC. Bars represent mean ±S.E.M, N=3 independent experiments. TNF-α ELISA revealed an increasing secretion of cytokine, TNF-αα with increasing LPS concentration

7 Products Seeding of human ipsc-derived microglial precursors The cells should be cultured for at least 14 days before use and then used for experiments within 7 days. Human ipsc-derived microglial precursors (ax1666) Human ipsc-derived microglial precursors; 3 million live cells per vial; Ready for assays in 14 days, ideal for creating co-culture of microglia and neurons for neuro-immunological studies and assay development. Axol s human ipsc-derived microglial precursors are generated using a fully defined medium, shipped live in our microglial maintenance medium. Three million cells are packed in each vial enough for plating 1 x 96-well plate. Human ipsc-derived microglia precursors will arrive warm at 37ºC in a cryovial. Gently pipette the microglia to get them into suspension and transfer the cells to a 15 ml conical tube (~ 2.4 ml). Wash the vial twice with 1 ml room temperature microglia maintenance medium (basal, non-supplemented) each time. Add a further 7.6 ml room temperature microglia maintenance medium to the 15 ml conical tube to make up to 10 ml. Centrifuge the cells at 300 x g for 5 minutes. Remove and discard the supernatant. Resuspend the cell pellet in room temperature microglia maintenance medium. Perform a cell count to determine total number of viable microglia precursors. ipsc-derived microglial precursors in microglia maintenance medium can be seeded directly onto tissue culture-treated plasticware. Axol recommends seeding at a density of 100,000 cells/cm2. Three million cells (1 vial) are enough for seeding 1 x 96-well plate. Incubate at 37ºC, 5% CO₂. Terminal differentiation to mature ipsc-derived microglia Pre-Shipment Post-Shipment Vial Cell Count Viability 1 4x % 2 4x % 2-day shipment Cell Count Viability 3.69x % 3.83x % After 14 days of differentiation and maturation in the microglial maintenance medium, the cells express key microgliaspecific markers, such as transmembrane protein 119 (TMEM119) and Purinergic Receptor P2Y12 (P2RY12), and myeloid markers TREM2 and IBA

8 Plated human ipsc-derived microglia (ax0666) Ready-to-use human ipsc-derived microglia in 96-well plate format (30,000 cells/well). Axol s human ipsc-derived microglia are generated using a fully defined medium from a factory of human ipsc-derived monocytes. These highly validated human ipsc-derived microglia have been successfully used in multiple applications to evaluate functionality such as assessment for cytokine secretion by ELISA, phagocytosis capability by phagocytosis and efferocytosis assays, and changes in the production of reactive oxygen species (ROS) by ROS assays. Human ipsc-derived microglia reveals the expression of key microglia-specific markers, such as transmembrane protein 119 (TMEM119) and Purinergic Receptor P2Y12 (P2RY12), and the myeloid markers TREM2 and IBA-1. Maintenance of human ipsc-derived microglia Axol s human ipsc-derived microglia are derived from ipscs which have undergone an intermediate differentiation to monocytes prior to directed terminal differentiation to microglia. The cells have undergone 14 days of microglia differentiation and can be used immediately for experiments if desired. Alternatively, they can be cultured for a maximum of 6 days further before use. The 96-well plate will arrive in a sealed plastic bag and the wells will be capped with a silicone seal. The human ipscderived microglia will be shipped on day 14 post differentiation from human ipsc-derived monocytes. The cells will arrive on day 15. Carefully open the plastic package and place the 96-well plate into a class II biosafety cabinet. Making sure to spray the plate with 70% ethanol and wipe it down. Remove the silicone sealing cap from the plate. Check cells under the microscope. Incubate the plate at 37ºC, 5% CO₂. To maintain a healthy microglia culture, gently replace 90 µl of medium with 100 µl of fresh, pre-warmed 37ºC, microglia maintenance medium every other day. Culture the human ipsc-derived microglia for a minimum of 1 day before conducting preferred assays. Optimally, microglia should be assayed within a few days of receiving the cells. Microglia maintenance medium kit (ax0660) Important! Axol Cell Culture Media DOES NOT contain antibiotics or antifungal agents. Axol Bioscience does not recommend the use of antimicrobial agents such as penicillin, streptomycin and amphotericin. Antimicrobial agents should not be necessary if proper aseptic technique is adopted. Axol s microglia maintenance medium is fully optimized to support the differentiation and culture of Axol s human ipsc-derived microglia (requires supplementing with the provided supplements A, B and C)

9 References 1. Karch, C. M. & Goate, A. M. Alzheimer s disease risk genes and mechanisms of disease pathogenesis. Biol. Psychiatry 77, (2015). Preparation of Reagents Supplement A 2. Morello, G. Spampinato, A. G. Cavallaro, S. Neuroinflammation and ALS: transcriptomic insights into molecular disease mechanisms and therapeutic targets. Mediators Inflamm (2017). 3. Crotti, A & Glass, C.K. The choreography of neuro inflammation in Huntington s disease. TrendsImmunol. 36, (2015). Store Supplement A at -80ºC on arrival. Remove the silicone sealing cap from the plate. After initial thaw, use 50 µl of Supplement A to make up 50 ml of microglia maintenance medium. Aliquot the remaining 50 µl and store at -80ºC until required. Do not thaw and refreeze more than once. Supplement B Store Supplement A at -80ºC on arrival. After initial thaw, use 50 µl of Supplement A to make up 50 ml of microglia maintenance medium. Aliquot the remaining 50 µl and store at -80ºC until required. Do not thaw and refreeze more than once. Supplement C Store Supplement A at -80ºC on arrival. After initial thaw, use 50 µl of Supplement A to make up 50 ml of microglia maintenance medium. Aliquot the remaining 50 µl and store at -80ºC until required. Do not thaw and refreeze more than once. Microglia Maintenance Medium Upon receipt, aliquot and store microglia maintenance medium at 4ºC. Microglia maintenance medium requires supplementing with Supplement A, B and C before use. Add the Supplements to the microglia maintenance medium according to the table. Once Supplements A, B and C have been added to the microglia maintenance medium it is stable for 1 week at 4ºC. Before use, pre-warm an aliquot of microglia maintenance medium at 37ºC

10 Frequently Asked Questions Do these cells come in frozen vials? No, these cells are shipped live in cryovials and must be plated immediately upon receipt. What is the purity of the microglia culture? Our microglia are ~95% pure, as assessed immunocytochemically by co-localisation of IBA-1 (myeloid marker) and P2RY12 (microglia-specific marker). See data on the product page at axolbio.com for further information. How do you ensure lot-to-lot consistency and what are the QC criteria? Microglia Precursors (ax1666) and required Medium Kit (ax0660) are tested for sterility and mycoplasma contamination. Cells must be >90% viable pre-shipment and test positive for the presence of IBA-1, CX3CR1, TMEM119 and P2RY12 after a 2-week differentiation to be considered as QC-passed. Is it possible to expand microglial precursors? No, the precursors are non-proliferative and will begin to differentiate upon attachment to cell-culture treated surfaces Can the microglia be detached and re-seeded? Once plated, the microglia adhere very strongly to cell culture-treated surfaces and are very difficult to detach without use of a cell scraper, which has the potential to cause mechanical damage and/or activate the cells. Passaging using Accutase with gentle scraping has been tested in-house, and while some cells do reattach, the ramified morphology is lost and does not recover. As such, we strongly recommend plating into final format. Can the cells be seeded onto glass and if so, is there a recommended coating? Microglial Precursors attach readily to glass slides/coverslips/wells and can be seeded directly onto untreated glass surfaces. Differentiation can be carried out for 2 weeks in maintenance medium as normal How long does shipping take on average? Usually 1-2 days depending on geographic location. In some circumstances, it may take up to 3 days. We have successfully tested a 3-day shipment of live microglial precursors with no detrimental effects to the cells or differentiation capacity. Notes How long can microglia be kept in good condition after 2 weeks of differentiation? A maximum of 1 week. After this, cells will begin to die off, which may affect downstream assays. How often are microglial precursors produced? Is it possible to specify a shipping date? The cells are made on a continuous rolling production, so should be readily available to purchase year-round. Each lot of cells produces for 4 weeks and vials can be reserved for shipment on a weekly basis over this period. Availability therefore depends on the number of reservations already placed for certain weeks of a given lot. Is the differentiation/maturation media provided? Accompanying media is sold separately as Microglia Maintenance Medium (ax0660), and its use specifies differentiation/ maturation for 2 weeks and subsequent maintenance of microglia for an additional 1 week maximum if desired. Are these cells similar to HSCs? These cells are quite different from blood-derived monocytes, in terms of ontogeny and gene expression profile. The generation of our ipsc-microglial precursors mimics the in vivo, myb-independent pathway of differentiation. Our cells are not HSC or HSC-like as these are by definition dependent on MYB for their self-renewal. That said, they express some classical BDM markers, being CD45+/CD11b+/CD14+ and can be differentiated to ipscmacrophages ( as well as ipsc-microglia. Differentiation to other lineages may be possible though it is at the purchaser s own risk

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