Generation of microglial cells from mouse embryonic stem cells

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nature protocols | VOL.5 NO.9 | 2010 | 1481

IntroDuctIonMicroglia are the resident immune cell type of the central nervous system (CNS) and constitute almost 10% of all brain cells1. They appear as ionized calcium-binding adaptor molecule-1 (Iba1)-positive cells within the neuroepithelium during early neuroecto-dermal development, before vasculature has developed2. Microglial precursors populating the CNS are most likely to be of mesodermal origin, although no formal evidence for this lineage exists2. In gen-eral, microgliosis under pathological conditions in adult mice is the result of a proliferative response by CNS-resident microglia3. Microglial cells contribute to a variety of important tasks, mainly related to the innate immune response. They are phagocytic cells responsible for the elimination of pathogens and apoptotic cells during tissue homeostasis. On stimulation by cytokines, such as interferon-γ (IFN-γ), microglia become activated and show an increased antigen-presenting capacity and increased synthesis of proinflammatory cytokines and toxic mediators1,4,5. Microglial function is often studied in primary microglial cells, which can be isolated and enriched from mixed glial cultures derived from brains of postnatal mice or rats. Alternatively, microglial cells can be directly isolated from adult brain tissue using density gradi-ents6 and flow cytometric sorting7. However, the yield obtained by isolating primary microglia in these ways is rather low. Therefore, many scientists instead use microglial cell lines, such as BV2 that are derived from primary microglia by oncogenic transformation8,9.

Recently, a protocol for differentiation of microglia-like cells from mouse embryonic stem (ES) cells has been described10. This study used a density-gradient method to isolate a sub-population of Iba1 + and CD45 + cells. The isolated cells had a microglial-like morphology after transplantation in mice, but were not described to proliferate and expand under culture conditions. In contrast, the protocol presented here leads to a stably proliferating microglial cell line. In our laboratory, we have generated several microglial precur-sor cell lines from mouse ES cells11 using the protocol described here, which is a modified version of a neuronal differentiation method12. We succeeded in obtaining ES cell-derived microglial precursor (ESdM) lines that were indistinguishable from primary microglia on the basis of their morphology and cell surface recep-tors11. ESdMs that differentiated from C57BL/6-ATCC ES cells

showed expression of markers that are essential for the confirma-tion of microglial status (including CD11b, CD11c, CD29, CD36, CD45, CD68, CD80, CD86, CD115 and Iba-1); however, stem cell markers CD34 or CD117, which are crucial for the elimination of remaining stem cells from the ESdM population, could not be detected. Throughout the protocol, we performed different immu-nostaining assays with antibodies directed against neural-specific marker proteins. After the neural differentiation step of the proto-col, we detected a large number of cells positive for the intermediate filament nestin. Thereafter, neuronal progenitor cells positive for β-tubulin III emerged, indicating a mixed neuronal cell population containing several cell types such as neuronal precursors and neu-rons. Within this mixed population, we immunostained cells with a microglia-like morphology with an antibody against the microglial actin-binding protein Iba1, the hematopoietic cell surface marker CD45 and the lysosome-associated membrane glycoprotein CD68. All three marker proteins are essential for determining microglial cells. By using an in vitro functional migration assay, we showed that ESdMs migrated toward the chemokine CX3CL1, indicating func-tional similarities of ESdM to primary microglial cells. We studied the capacity for phagocytosis of ESdMs by the uptake of micro-sphere beads13. We showed that ~20% of ESdMs phagocytosed at least two microsphere beads. The percentage of ESdMs showing bead uptake increased on cell stimulation with lipopolysaccharides (LPS). Furthermore, ESdMs engraft as microglial cells into brain tissue on transplantation11 and have been applied as vehicles for CNS gene therapy in an animal model of multiple sclerosis 14.

The results obtained from ESdMs with respect to surface marker expression or migratory and phagocytic functions have been stably reproduced up to passage 25 (ref. 11). Because damage mediated by oxidative stress might accumulate in long-term cell cultures, ESdMs should not be used at higher passages. Thus, ESdMs provide a cellu-lar tool to study the function of microglia in vivo and in vitro with-out oncogenic transformation or limited cell number. In addition, the protocol described here could be applied to a broad range of ES cells derived from transgenic mice, leading to genetically modi-fied ESdMs, which further broadens the possibilities for analyzing microglial function and their involvement in CNS diseases.

Generation of microglial cells from mouse embryonic stem cellsClara Beutner, Kristin Roy, Bettina Linnartz, Isabella Napoli & Harald Neumann

Neural Regeneration Group, Institute of Reconstructive Neurobiology, University of Bonn, Bonn, Germany. Correspondence should be addressed to H.N. ([email protected]).

Published online 5 August 2010; doi:10.1038/nprot.2010.90

Microglia, the resident immune cells of the brain, are difficult to obtain in high numbers and purity using currently available methods; to date, microglia for experimental research are mainly isolated from the brain or from mixed glial cultures. In this paper, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem (es) cells into microglial precursor cells. Microglia are obtained by a protocol consisting of five stages: (i) cultivation of es cells, (ii) formation and differentiation of embryoid bodies, (iii) differentiation into neuroectodermal lineage and isolation of myeloid precursor cells, (iv) differentiation into microglial precursor cells and (v) cultivation of es cell-derived microglial precursors (esdMs). the protocol can be completed in 60 d and results in stably proliferating esdM lines, which show inducible transcription of inflammatory genes and cell marker expression comparable with primary microglia. Furthermore, esdMs are capable of chemokine-directed migration and phagocytosis, which are major functional features of microglia.

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1482 | VOL.5 NO.9 | 2010 | nature protocols

Overview of the procedureEmbryonic stem cell-derived microglial precursors should provide a reliable source of cells for replacing primary microglia or onco-genically transformed microglial cell lines. The aim of the protocol presented here is to generate ESdMs from mouse ES cells (after ES cells are tested for mycoplasma contamination and frozen in liquid nitrogen) using a relatively easy and effective five-step procedure as outlined below:

Culture of ES cells: ES cells are expanded on a mouse embryonic fibroblast (MEF) layer in ES medium in the presence of leukemia inhibitory factor (LIF) to prevent differentiation.Induction of embryoid body formation: ES cells are trypsinized and transferred to gelatin-coated dishes for 1 d before being transferred to nonadherent Petri dishes in ES medium without LIF. This step induces differentiation and formation of aggregates.Neural differentiation: At 4 d after embryoid body formation, cells are replated on gelatin-coated tissue culture dishes. After 2 d, the medium is changed to ITSFn medium, which supports expansion of nestin-positive cells.Microglial differentiation: After 6 d, the medium is changed to N2 medium supplemented with fibroblast growth factor-2 (FGF2) and laminin to enhance microglial differentiation and expansion. In the final step of the differentiation (day 26), growth factors are removed. After 3 weeks, stably proliferating ESdMs are visible. To purify the ESdMs, single cell colonies of ESdMs can be picked manually using a micropipette.Culture of ESdMs: ESdMs are cultured in serum-free N2 medium. They are split using a cell scraper when they are 80% confluent.

The protocol presented here for differentiation of mouse ES cells into microglia was established using C57BL/6-ATCC ES cells. We have successfully applied the protocol, without modification, to sev-eral ES cell lines derived from a variety of knockout mice. However,

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longer periods of differentiation may be required to obtain ESdMs from genetically modified ES cells (see Step 55 of the Procedure). Furthermore, we cannot exclude the possibility that some sources of ES cells might not be suitable for the protocol. In particular, ES cells lacking genes essential for myeloid differentiation might require modification of the protocol or addition of specific growth factors.

Future directionsThis protocol describes the generation of a microglial precursor cell line, which will differentiate and polarize into microglial cells after integration into the CNS tissue depending on environmental cues and the micromilieu11. It is well known that microglia can have either cytotoxic or regenerative effects depending on their stage of polarization. It is believed that this two-edged nature of micro-glia is a consequence of polarization in nitric oxide-producing or phagocytically active microglia. For macrophages, it is possible to trigger regenerative polarization in vitro using cytokines such as interleukin 4 or interleukin 10 (ref. 15), whereas the classical activation for cytotoxic polarization can be performed using IFN-γ or other proinflammatory mediators such as LPS16. The generated ESdM lines strongly express several endocytic and phagocytic receptors such as TREM2 and CD163 and might reflect a transition stage between embryonic myeloid cells and microglia, indicating microglial precursors that populate the CNS during development17. Ongoing studies will reveal whether ESdMs can be polarized in vitro to study effector function of matured microglia in more detail. Furthermore, generation of microglia from human ES cells or induced pluripotent stem cells is of prospective interest.

MaterIalsREAGENTS crItIcal The reagents used in this protocol do not have to be purchased from the suppliers mentioned. It is very likely that alternative products can be used without any changes in the differentiation capacity. However, we have not tested other products and thus recommend the use of the reagents listed below to ensure the success of the differentiation protocol.

2-Mercaptoethanol (50 mM, 1,000×; Gibco, cat. no. 31350010) ! cautIon It is toxic and hence avoid exposure; use it under a fume hood.4,6-Diamidino-2-phenylindol (DAPI; Sigma, cat. no. D9542)Antibodies (see Tables 1–4)

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table 1 | List of primary antibodies for immunocytochemistry.

antibody HostDirected against Dilution

supplier, cat. no.

β-Tubulin-III Mouse Neurons 1:500 Sigma, T8660

CD45 Rat Leukocytes 1:500 BD Pharmingen, 550539

CD68 Rat Monocytes, macrophages

1:500 AbD Serotec, MCA1957

GFAP Rabbit Astrocytes 1:1,000 DAKO, Z0334

Iba1 Rabbit Microglia 1:500 or 1:2,000

Wako, 019-19741

Nestin Mouse Neural marker, neuronal stem cells

1:200 Millipore, MAB353

Bovine serum albumin (BSA; Sigma, cat. no. A4503-100G; see REAGENT SETUP)C57BL/6-ATCC ES cells (ATCC, cat. no. SCRC-1002)CD-1 mice (Charles River) ! cautIon Experiments on animals must be performed according to facility-approved guidelines and regulations.CX3CL1 (obtained from recombinant mouse; R&D Systems, cat. no. 472-FF; see REAGENT SETUP)d-Glucose solution (45% (wt/vol); Sigma, cat. no. G8769)DIFF medium (see REAGENT SETUP)DABCO (1,4 diazabicyclo-[2,2,2]-octane; Sigma, cat. no. D2522)DMEM with 4.5 g per liter d-glucose (Gibco, cat. no. 41965)DMEM/F12 (Gibco, cat. no. 31330)DMSO (Sigma, cat. no. D8418)DPBS (Dulbeco’s PBS; Gibco, cat. no. 14190)ES medium (see REAGENT SETUP)Fetal calf/bovine serum (FCS; Gibco, cat. no. 10270; see REAGENT SETUP)FGF2 (obtained from recombinant human; R&D Systems, cat. no. 233-FB; see REAGENT SETUP)Fibronectin (obtained from bovine plasma; Sigma, cat. no. F4759; see REAGENT SETUP)Fluoresbrite polychromatic red (microspheres, 1.0 µm; Polyscience, cat. no. 18660-5)Gelatin (Fluka, cat. no. 04055; see REAGENT SETUP)GlutaMAX (200 mM; Gibco, cat. no. 35050)Glycerol (Sigma, cat. no. G7757)Granulocyte macrophage-colony stimulating factor (GM-CSF, obtained from recombinant mouse; Invitrogen, cat. no. PMC2014; see REAGENT SETUP)IFN-γ (obtained from recombinant mouse; R&D Systems, cat. no. 485-MI; see REAGENT SETUP)Insulin (Sigma, cat. no. I6634; see REAGENT SETUP)ITSFn medium (see REAGENT SETUP)Laminin (obtained from natural mouse; Invitrogen, cat. no. 23017-015; see REAGENT SETUP)

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Leukemia inhibitory factor (LIF, obtained from recombinant mouse; Chemicon, cat. no. LIF2010)l-Glutamine (200 mM, 100×; Gibco, cat. no. 25030)Lipopolysaccharides (LPS; Sigma, cat. no. L5886; see REAGENT SETUP)LookOut Mycoplasma PCR detection kit (Sigma, cat. no. MP0035)MEF medium (see REAGENT SETUP)MEM nonessential amino acid solution (100×; Gibco, cat. no. 11140)Moviol 4-88 (Kremer Pigmente, cat. no. 67760; see REAGENT SETUP)N2 medium (see REAGENT SETUP)N2 supplement (100×; Gibco, cat. no. 17502048)NaOH (1 M; Roth, cat. no. K021.1)Penicillin/streptomycin (pen/strep; Gibco, cat. no. 15140163)Paraformaldehyde (PFA; Merck, cat. no. 104005; see REAGENT SETUP) ! cautIon PFA is a formaldehyde-releasing agent and could be a carcinogen; harmful if inhaled! Use it under a fume hood.RNeasy mini kit (Qiagen, cat. no. 74106)MEM sodium pyruvate solution (100 mM; Gibco, cat. no. 11360)Sodium selenite (Sigma, cat. no. S5261; see REAGENT SETUP)SuperScript III platinum two-step qRT-PCR kit (Invitrogen, cat. no. 11734050)SYBR Green master mix (2×; Applied Biosystems, cat. no. 4309155 )Tris buffer (0.2 M, pH 8.5; Roth, cat. no. 54293)TritonX-100 (Sigma, cat. no. T9284)Transferrin bovine (Sigma, cat. no. T-0178; see REAGENT SETUP)Oligonucleotides (for qRT-PCR; Eurofins MWG)Trypsin-EDTA (0.25% (wt/vol); Gibco, cat. no. 25200)

EQUIPMENTStericup (0.22 µm pore size; Millipore, cat. no. SCGPT05RE)Tissue cell culture flask (175 cm2; Sarstedt, cat. no. 83.1812.002)Disposable plastic pipette (5 ml; Costar, cat. no. 4487)Six-well plate (Cellstar, cat. no. 657160)Culture plate inserts (8.0-µm, 12-mm diameter; Millipore, cat. no. PI8PO1250)Disposable plastic pipette (10 ml; Costar, cat. no. 4488)Tubes (15 ml; Greiner, cat. no. 188271)Twenty-four-well plate (Greiner bio-one, cat. no. 662102)Disposable plastic pipette (25 ml; Costar, cat. no. 4489)Plastic tubes (50 ml; Sarstedt, cat. no. 62.547.254)Axioskop 2 (Zeiss)Axiovert 40 CFL (Zeiss)BD FacsCalibur (BD Biosciences)Canon Powershot G9 (Canon)Cell scraper (Sarstedt, cat. no. 83.1830)Cell strainer (BD Falcon, cat. no. 352340)Cotton buds (Roth, cat. no. EH11.1)Eppendorf mastercycler epgradient S (Eppendorf)Erlenmeyer flask (250 ml, Schott-Duran, cat. no. 21 217 36)Glass beads (5 mm; Roth, cat. no. HH56.1)

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table 2 | List of secondary antibodies for immunocytochemistry.

antibody HostDirected against Dilution supplier, cat. no.

Alexa488 Goat Anti-mouse 1:500 Invitrogen, A31619

Alexa488 Goat Anti-rabbit 1:500 Invitrogen, A11070

Alexa488 Goat Anti-rat 1:500 Invitrogen, A11006

Cy3 Goat Anti-rabbit 1:500 Dianova, 111-165-047

Cy3 Goat Anti-mouse 1:500 Dianova, 115-166-072

Cy3 Goat Anti-rat 1:500 Dianova, 112-166-072

Glass cover slides (24 × 24 mm; VWR, cat. no. 631-1570)Glass Pasteur pipettes (Brand, cat. no. 747715)HeraCell 240 incubator (Heraeus)Lab-Tek chamber slide w/cover permanox slide sterile four-well (Labomedic, cat. no. 177437)MicroAmp optical adhesive film (96- and 384-well; Applied Biosystems, cat. no. 4311971)MicroAmp optical plate (96-well; Applied Biosystems, cat. no. 4306737)Neubauer chamber (Brand, cat. no. 718605)Petri dishes (100×15 mm; BD Falcon, cat. No. 351029)RS 2000 X-ray source (Rad Source Technologies) ! cautIon Radiation causes cancer and DNA damage. Always be aware of regulations and wear appropriate personal protective equipment.Surgical instruments for MEF preparation, variousTissue culture dish (100×20 mm; Sarstedt, cat. no. 83.1802.003)Tissue culture dish (60×15 mm; Sarstedt, cat. no. 83.1801.003)Tissue culture dish (150×20 mm; TPP, cat. no. 93150)Tubes (1.5 ml; Eppendorf, cat. no. 3810)

REAGENT SETUPBSA (1%) Prepare 100 ml stock solution using the sterile 10% (wt/vol) BSA solution (1% (wt/vol) in sterile DPBS), store 10 ml aliquots at -18 °C for up to 1 year.BSA (1% (wt/vol)) Prepare 100 ml stock solution (10% (wt/vol) in sterile DPBS), filter-sterilize with Stericup filter and store 10 ml aliquots at -18 °C for up to1 year.CX3CL1 Prepare 1 ml sterile stock solution of 25 µg ml − 1 in 1% BSA in DPBS. Store 20-µl aliquots at − 20 °C for up to 3 months.DAPI Prepare a stock solution of 0.5 mg ml − 1 in water, store in 20-µl aliquots at − 20 °C for up to 1 year. For staining, dilute 1:5,000 in DPBS.DIFF medium DIFF medium is composed of DMEM (with 4.5 g per liter of d-glucose) supplemented with 2 mM GlutaMAX, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.05 mM 2-mercaptoethanol and 15% (vol/vol) FCS.

To prepare 500 ml of DIFF medium, combine 5 ml of GlutaMAX, 5 ml of sodium pyruvate, 5 ml of nonessential amino acids, 5 ml of 2-mercaptoethanol and 75 ml of FCS; adjust the volume to 500 ml with DMEM (with 4.5 g per liter of d-glucose), filter sterilize and store at 4 °C. Use within 2 weeks.ES medium ES medium is composed of DMEM (with 4.5 g per liter of d-glucose) supplemented with 2 mM GlutaMAX, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.05 mM 2-mercaptoethanol, 15% (vol/vol) FCS and 1 µg per liter LIF.

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table 3 | List of primary antibodies for flow cytometry.

antibody Host Directed against Dilution supplier, cat. no.

CD11b-bio Rat Granulocytes, monocytes, NK cells and tissue macrophages

1:200 BD Pharmingen, 553309

CD11c-bio Hamster Monocytes, macrophages, neutrophils

1:200 BD Pharmingen, 553800

CD16/32 Rat Fc-receptor blockade 1:100 BD Pharmingen, 553142

CD29 Rat Leukocytes 1:200 BD Pharmingen, 553731

CD34 Rat Stem cells 1:200 BD Pharmingen, 550537

CD36-bio Mouse Monocytes 1:200 BD Pharmingen, 552544

CD45-bio Rat Leukocytes 1:200 BD Pharmingen, 553078

CD80-bio Rat B cells, monocytes 1:200 BD Pharmingen, 553767

CD86-bio Rat T cells 1:200 BD Pharmingen, 553690

CD115-bio Sheep Monocytes, macrophages 1:40 R&D Systems, BAF3818

CD117/cKit-bio Rat Stem cells 1:200 BD Pharmingen, 553353

Isotype IgG1 Rat 1:200 BD Pharmingen, 553922

Isotype IgG2 Rat 1:200 BD Pharmingen, 553980

Isotype IgA Mouse 1:200 BD Pharmingen, 553476

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To prepare 500 ml of ES medium, combine 5 ml of GlutaMAX, 5 ml of sodium pyruvate, 5 ml of nonessential amino acids, 5 ml of 2-mercaptoethanol, 75 ml of FCS and 50 µl of LIF; adjust the volume to 500 ml with DMEM (with 4.5 g per liter of d-glucose), filter-sterilize and store at 4 °C. Use within 2 weeks.FCS Store FCS at − 18 °C. Before use, thaw, heat immobilize at 56 °C for 30 min, prepare aliquots (50 ml) and refreeze at − 18 °C. Aliquots are stable up to the manufacturer’s expiration date of the unheated FCS. Only use heat-immobilized aliquots for the protocol.FGF2 Prepare a stock solution of 1 µg ml − 1 using sterile DPBS with 0.1% BSA. Store 10-µl aliquots at − 80 °C; the aliquots stable for 3 months.Fibronectin Prepare a stock solution of 5 mg ml − 1 in DPBS and incubate for 30 min at 37 °C. Store 20-µl aliquots at − 18 °C; the aliquots are stable for 6 months.Gelatin (0.1% (wt/vol) solution) Dissolve 1 g of gelatin powder in 1 liter of distilled water, filter sterilize and autoclave. Store at room temperature (RT, 20 °C) for up to 2 months. To coat dishes, apply an appropriate volume of gelatin solution (~10 ml) to cover the entire bottom of a dish and incubate for 30 min at 37 °C. Aspirate solution before using dishes.GM-CSF Prepare a stock solution of 20 µg ml − 1 with sterile water. Aliquots of 20 µl can be stored at − 80 °C for up to 1 year.IFN-γ Prepare a sterile stock solution of 1,000,000 U ml − 1 in DPBS and store at –80 °C for up to 1 year.Insulin Prepare a stock solution of 25 µg ml − 1 with sterile water. Aliquots (500 µl) are stored at − 80 °C and are stable for 1 year.ITSFn medium ITSFn medium is composed of DMEM/F12 supplemented with 25 µg ml − 1 insulin, 30 nM sodium selenite and 50 µg ml − 1 transferrin. Add 5 µg ml − 1 of fibronectin fresh before using the medium.

To prepare 500 ml of ITSFn medium, combine 500 µl of insulin, 30 µl of sodium selenite and 2.5 ml of transferrin. Add 10 µl of fibronectin freshly to each 10 ml of medium. Filter sterilize and store at 4 °C for up to 2 weeks.Laminin Thaw laminin (1 mg ml − 1) and prepare aliquots of 20 µl. Store at − 20 °C for up to 6 months.LPS Prepare 5 ml of sterile stock solution of 1 mg ml − 1 in DPBS. Store at − 20 °C for up to 2 years.MEF culture preparation Generated from CD-1 mice according to procedures described in reference 18 (see Box 1).MEF medium MEF medium is composed of DMEM (with 4.5 g per liter of d-glucose) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids and 10% (vol/vol) FCS.

To prepare 500 ml of MEF medium, combine 5 ml of l-glutamine, 5 ml of sodium pyruvate, 5 ml of nonessential amino acids and 50 ml of FCS; adjust the volume to 500 ml with DMEM (with 4.5 g per liter of d-glucose), filter-sterilize and store at 4 °C for up to 2 weeks.Moviol Dissolve 4.8 g of Moviol and 12 g of glycerol in 12 ml of sterile water and incubate for 3 h at RT. Add 24 ml of Tris buffer and stir for 15 min at 50 °C. Add 1.32 g of DABCO. Prepare 1-ml aliquots and store at − 20 °C for up to 1 year.N2 medium N2 medium is composed of DMEM/F12 supplemented with 1× N2 supplement, 0.48 mM l-glutamine, 5.3 µg ml − 1 d-glucose and 100 µg ml − 1 pen/strep solution.

To prepare 500 ml of N2 medium, combine 5 ml of N2 supplement, 1.2 ml of l-glutamine, 1.7 ml of d-glucose (45%) and 5 ml of pen/strep solution; adjust the volume to 500 ml with DMEM/F12. Filter-sterilize and store at 4 °C for up to 2 weeks. During microglial differentiation step, add 1 µg ml − 1 of laminin (10 µl of stock solution to 10 ml of medium) and 10 ng ml − 1 of FGF2 (10 µl of stock solution to 10 ml of medium) fresh to the dishes.PFA (4% (wt/vol)) Add 20 g of PFA to 400 ml of sterile water and heat to 65 °C. Add NaOH until the solution clears, then add 50 ml of 10× DPBS and cool on ice. Adjust the pH to 7.3 and make up the volume to 500 ml with sterile water. Prepare 50 ml aliquots and store at − 20 °C for a maximum of 6 months.Sodium selenite Prepare 1 ml of stock solution of 500 µM with sterile water. Store at 4 °C for up to 1 year.Transferrin Prepare a stock solution of 10 mg ml − 1 with sterile water. Store aliquots (2.5 ml) at − 80 °C for a maximum of 1year.

table 4 | List of secondary antibodies for flow cytometry.

antibody Host Directed against Dilution supplier

PE Streptavidin 1:500 Jackson ImmunoResearch, 016-110-084

PE Goat Anti-rat 1:500 Jackson ImmunoResearch, 112-116-143

proceDureMeF culture ● tIMInG 60 min1| Precoat a 10-cm tissue culture dish with gelatin for 30 min at 37 °C. (see REAGENT SETuP)

2| Prewarm MEF medium in a 37 °C water bath.

3| Working in a sterile hood, aspirate gelatin from the 10-cm culture dish (from Step 1) using a glass pipette.

4| Remove a vial of irradiated MEF cells (~4,000,000 cells) from storage in liquid nitrogen. Place the vial into the 37 °C water bath until the cells are thawed.

5| under a sterile hood, transfer the thawed cells into 10 ml of prewarmed (37 °C) MEF medium in a 15-ml tube.? troublesHootInG

6| Centrifuge for 3 min at 300g at RT.

7| Aspirate the supernatant with a glass pipette and resuspend the cells in 10 ml of prewarmed (37 °C) MEF medium.

8| Transfer cells to gelatin-coated dish (from Step 3) incubate overnight at 37 °C in 5% CO2. crItIcal step Make sure to have a dish of MEF cells ready for passaging the ES cells. MEF culture dishes can be kept up to 1 week while changing the MEF medium every third day. The MEF cells must cover at least 70% of the dish and should be well attached to the dish before the ES cells are added.? troublesHootInG

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es cell thawing ● tIMInG 30 min9| Prewarm ES medium in a 37 °C water bath.

10| Remove a vial of frozen, mycoplasma-free ES cells from storage in liquid nitrogen and place it into the 37 °C water bath until cells are thawed. crItIcal step We recommend performing mycoplasma tests with the LookOut Mycoplasma PCR Detection Kit (see REAGENTS) on a regular basis (every other week).

11| Transfer the cells under sterile conditions into 10 ml of prewarmed (37 °C) ES medium in a 15-ml tube.


13| Aspirate the supernatant with a glass pipette and resuspend the cells in 10 ml of ES medium.

14| Aspirate MEF medium from a dish containing MEF cells (from Step 8) and transfer ES cells to this dish. Incubate at 37 °C, 5% CO2.

Box 1 | MEF CULTURE PREPARATIoN ● tIMInG 30 d Day 1: 1. Set up a breeding pair of CD-1 mice.Day 2: 2. To assess pregnancy, check for vaginal plugs on females and separate those with plugs (pregnant) from those without.Day 16: 3. Prepare feeder cells. Thereafter, kill pregnant mice and rinse well with 70% (vol/vol) ethanol. 4. under a sterile hood, isolate embryos and wash several times with DPBS. Transfer them into a dish containing cold DPBS. 5. using sterile forceps, disrupt the embryonic sac and free the embryo. Remove the fetal liver and heart. Place embryos in a new dish

containing cold DPBS. 6. Remove the heads of the embryos and place the decapitated mouse embryos in a new dish with cold DPBS. 7. Mince the embryos with scissors. Transfer minced tissues together with 50 ml of trypsin-EDTA into an Erlenmeyer flask containing

5-mm sterile glass beads. Stir very gently at 37 °C for 30 min. 8. Depending on the number of embryos used, add MEF medium to the flask from Step 7 and transfer to 50-ml tubes. Wash the

remaining glass beads with MEF medium and transfer to 50-ml tubes. Centrifuge at 300g for 10 min at RT. 9. Resuspend pellet in medium and count the cells with a Neubauer chamber. 10. Seed cells in 15-cm cell culture dishes at a minimum of 5,000,000 cells in 20 ml of prewarmed (37 °C) MEF medium; culture at

37 °C in 5% CO2.11. upon confluency, freeze cells in MEF medium with 10% (vol/vol) DMSO. Freeze ~4,000,000 cells per vial.12. When needed, thaw 1 vial of MEF cells in the water bath (37 °C). Wash the cells by adding 10 ml of prewarmed (37 °C) MEF medium,

centrifuging at 300g for 3 min at RT and aspirating the medium with a glass pipette. Resuspend pellet in 6 ml of MEF medium. 13. Add 1 ml of the cell suspension to one 15-cm tissue culture dish each. Add 19 ml of prewarmed MEF medium and incubate at

37 °C in 5% CO2. Throughout the culture, change medium every third day.14. upon confluency, split cells: Aspirate medium with a glass pipette, wash once by adding 10 ml of prewarmed (37 °C) DPBS and

aspirating it, and add 10 ml of trypsin-EDTA. Wait until cells detach, and then transfer the cell solution into 50-ml tubes containing 20 ml of prewarmed MEF medium each. Centrifuge for 5 min at 300g at RT. Resuspend pellet in 18 ml of MEF medium and distribute to 18 new 15-cm dishes. Add 19 ml of prewarmed MEF medium to each dish.

15. upon confluency, repeat Step 14, splitting the cells into 54 dishes.16. upon confluency, detach cells with trypsin-EDTA (see Step 14).17. Collect cell suspension in 50 ml tubes containing 20 ml prewarmed (37 °C) MEF medium. Centrifuge at 300g for 5 min at RT.

Resuspend the pellets in 1 ml of cold MEF medium. 18. Collect equal volumes of cell suspension in two 50-ml tubes and fill up to 40 ml with cold MEF medium. Transfer the solutions to

two 175-cm2 cell culture flasks on ice.19. Irradiate with 15 Gy using a cesium source for irradiation (for example, the RS 2000 X-ray source from Rad Source Technologies).

! cautIon Please take appropriate precautions when working with radiation. 20. Collect suspension in 50-ml tubes and centrifuge at 300g for 5 min at 4 °C. 21. Resuspend in cold MEF medium and count cells using a Neubauer chamber. 22. Adjust to ~3,000,000 cells per ml in 40% MEF medium, 50% FCS and 10% DMSO. Slowly freeze 1 ml per vial. Store in liquid

nitrogen; use within 2 years.

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es cell expansion ● tIMInG 10 min every day for 2–3 d15| Change ES medium every day. Aspirate old medium with a glass pipette and replace with 10 ml of prewarmed (37 °C) ES medium until the cells are 60% confluent (2–3 d), then split them as described in Steps 16–22 below.? troublesHootInG

es cell passaging ● tIMInG 30 min16| Prewarm DPBS, trypsin and ES medium separately to 37 °C.

17| Aspirate old medium from the ES cell culture dish with a glass pipette, and wash once by adding DPBS and then aspirating it from the dish.

18| Add 5 ml of trypsin and incubate for 1 min at 37 °C in 5% CO2.

19| Pipette up and down several times with a 5-ml plastic pipette until all the cells are detached, then transfer cells to 5 ml of prewarmed (37 °C) ES medium in a 15-ml tube.

20| Incubate for 5 min until about 70% of the MEF cells are sedimented. Transfer upper 5 ml of the solution to fresh tube and fill up to 10 ml with prewarmed (37 °C) ES medium.


22| Resuspend the cells in 10 ml of prewarmed (37 °C) ES medium and split them 1:5 to 1:10 (by transferring a fifth or a tenth of the 10-ml suspension) to a fresh MEF dish. Adjust to 10 ml with prewarmed (37 °C) ES medium. crItIcal step Make sure that the confluence of the ES culture does not exceed 60% or the cells will start to differentiate.

Induction of embryoid body formation ● tIMInG day 0–40 min23| Precoat tissue culture dish with gelatin for 30 min (see REAGENT SETuP)

24| Resuspend 1 ml of ES cell suspension obtained while passaging (Step 22) in 10 ml of prewarmed (37 °C) ES medium and transfer to gelatin-coated tissue culture dish. Incubate for 1 d at 37 °C in 5% CO2.

Formation of embryoid bodies ● tIMInG 30 min on day 1; 40 min on day 225| Day 1: Prewarm DPBS, trypsin and DIFF medium separately to 37 °C.

26| Aspirate ES medium from the tissue culture dish (from Step 24) with a glass pipette, and wash once by adding DPBS and then aspirating it.

27| Add 5 ml of trypsin and incubate for 1 min at 37 °C in 5% CO2.

28| Pipette up and down several times with a 5-ml plastic pipette until all cells are detached; transfer cells to 5 ml prewarmed (37 °C) DIFF medium in a 15-ml tube.

29| Incubate for 5 min to sediment the remaining MEF cells (from Step 20). Transfer upper 5 ml of the solution to a fresh tube and fill up to 10 ml with prewarmed (37 °C) DIFF medium.


31| Resuspend the cells in 10 ml of DIFF medium, transfer to a Petri dish and incubate for 1 d at 37 °C in 5% CO2.

32| Day 2: Prewarm DIFF medium to 37 °C.

33| use a 10-ml plastic pipette to pipette cell suspension several times very carefully up and down.

34| Transfer cell suspension to a 15-ml tube and incubate for 5 min at RT; embryoid bodies should sediment.

35| Aspirate upper 5 ml with a glass pipette and add 5 ml of the warm DIFF medium (from Step 32).

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36| Transfer cell suspension to a new Petri dish and incubate for 3 d at 37 °C in 5% CO2.? troublesHootInG

neural differentiation ● tIMInG 30 min on day 5; 10 min on days 7, 9, 11, 13; 10 min per d on days 14–2137| Day 5: Prewarm DIFF medium to 37 °C.

38| Transfer embryoid bodies to a 15-ml tube, incubate for 5 min until embryoid bodies are sedimented.

39| Remove upper 5 ml with a glass pipette and refill with fresh DIFF medium (from Step 37).

40| Plate embryoid bodies that are visible with the naked eye by transferring them to a tissue culture dish precoated with gelatin. Incubate for 2 d at 37 °C in 5% CO2. Plate no more than 20 embryoid bodies on a 10-cm dish or the outgrowing cells of the plated EBs might become too dense for the differentiation protocol. Consider plating at least three extra dishes for staining procedures.

41| Day 7: Prewarm ITSFn medium and DPBS to 37 °C.

42| Wash cells once by adding 10 ml of DPBS and aspirating it with a glass pipette.

43| Add 10 ml of ITSFn medium (add 10 µl of fibronectin fresh to each 10 ml of medium; it should be added to the fresh medium in the dish) to the cells and incubate at 37 °C in 5% CO2. Fibronectin is crucial for enhanced cell attachment and differentiation.

44| Change ITSFn medium every 48 h.

45| Carry out immunostaining for nestin between days 13 and 16 using an additional dish from Step 40 (see box 2).

46| Day 14: Prewarm DPBS and N2 medium separately to 37 °C.

47| Wash cells once by adding 10 ml DPBS and aspirating it with a glass pipette.

48| Add 10 ml of N2 medium and 10 µl each of laminin and FGF2 to the cells and incubate at 37 °C in 5% CO2. Laminin and FGF2 are added to the culture to further differentiate the neuronal precursor cells.

49| Change medium every other day until day 21. Add laminin and FGF2 every day.

Microglial differentiation ● tIMInG 10 min on day 21 and every other day thereafter50| Prewarm DPBS and N2 medium to 37 °C.

Box 2 | IMMUNoCYToCHEMISTRY ● tIMInG 50 MIN oN dAY 1; 1 H 20 MIN oN dAY 2 Day 1: 1. Wash cells (on extra dishes from Step 40) three times with 10 ml of DPBS. Note that staining can be performed under non-sterile conditions. 2. Fix cells with 5 ml of 4% PFA (see REAGENT SETuP) for 10 min. 3. Wash three times with 10 ml DPBS by adding PBS and aspirating it immediately. 4. Block for 30 min with 5 ml DPBS containing 10% (wt/vol) BSA for co-staining of Iba1 and CD68. For all other antibodies, block

with DPBS containing 10% (wt/vol) BSA, 5% (vol/vol) normal goat serum (because none of the primary antibodies used is raised in goats) and 0.1% (vol/vol) Triton X-100; aspirate.

5. Add primary antibodies (see table 1) in DPBS with 1% (wt/vol) BSA for co-staining of Iba1 and CD68 or DPBS with 5% (vol/vol) normal goat serum (from the previous step) for all other primary antibodies and incubate overnight at 4 °C.

Day 2: 6. Wash cells three times for 5 min with DPBS. 7. Add secondary antibodies in DPBS and incubate for 2 h for co-staining of Iba1 and CD68. Incubate all other secondary antibodies

in the dark for 30 min. 8. Wash three times for 5 min with DPBS. 9. Stain with DAPI (1:5,000 in DPBS) for 30 s.10. Wash three times for 5 min with DPBS.11. Cover with Moviol and glass cover slides and store at 4 °C in the dark.12. Analyze slides using a fluorescence microscope (Axioskop 2, Zeiss) or confocal microscope (Fluoview FV1000, Olympus).

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51| Wash cells (from Step 49) once by adding 10 ml DPBS and aspirating it with a glass pipette.

52| Add 10 ml of N2 medium and 10 µl of laminin to the cells. Incubate at 37 °C in 5% CO2.

53| Change medium and add laminin every other day. From day 26 on, stop adding laminin. The withdrawal leads to microglial differentiation of the neuronal cell culture.

54| Day 30: Carry out immunostaining for Iba1, CD45, β-tubulin III and GFAP using an additional dish from Step 40 (see box 2).

55| Approximately 3 weeks after growth factor withdrawal, the first microglial cells should appear. At this time point, it is an option to add 10 µl of GM-CSF to the cells with every medium change to improve the proliferation of ESdMs.

56| After 4 weeks, isolate the developed ESdM colonies using either option A or option B. Option A will lead more quickly to a purified culture containing less cells. Option B takes about 2 weeks longer, but results in a higher cell number.(a) Manual selection of colonies (i) Pick the ESdM colonies manually using a pipette tip. (ii) Transfer all ESdM colonies (up to 20 per culture dish) to a fresh 6-cm PLL-coated tissue culture dish in N2 medium. (iii) Wait 2 more weeks for other cell types to die, thereby obtaining a population of high purity.(b) non-manual selection of colonies (i) Wait 2 more weeks until the ESdMs start to outgrow other cell types; these other cell types typically will disappear at

this time point. ? troublesHootInG

culture of esdMs ● tIMInG 10 min every other day, 30 min every fourth day57| Culture ESdMs in N2 medium on PLL-coated tissue culture dishes, which results in a pure ESdM culture. Change medium every 48 h.? troublesHootInG

58| Immunostain one dish of ESdMs for Iba1 and CD68 (see box 2) when they are stably proliferating (day 50 or later).

59| When cells reach 80% confluence (3–4 d), split the cells in a ratio of 1:5 into PLL-coated tissue culture dishes as described below in Steps 60–62.

60| Prewarm N2 medium to 37 °C.

61| For splitting, wash cells once with 10 ml of DPBS and aspirate with a glass pipette.

62| Add 5 ml of medium to the cells and scrape them with a cell scraper.

63| Transfer 1 ml of the cell suspension to a new dish and add 9 ml of prewarmed (37 °C) N2 medium. pause poInt ESdMs can be frozen slowly in N2 medium supplemented with 10% DMSO and 40% FCS at a density of 4,000,000 cells per ml.? troublesHootInG

characterization of esdMs 64| The following experiments are suited for characterization of the ESdM culture. The migration assay (option A), the phagocytosis assay (option B) and the real-time quantitative PCR (qRT-PCR) (option D) are performed to determine the functional capacity of the ESdMs. Flow cytometry (option C) and immunocytochemistry (see box 2) are performed to analyze the surface marker expression. With the exception of immunocytochemistry, which should be performed at the indicated time points in the protocol, the order in which experiments are done does not matter. However, we recommend performing all the listed experiments to be able to fully reveal the functional capacity and markers of the obtained ESdMs.(a) Migration assay ● tIMInG 7–8.5 h (i) Prewarm DPBS and N2 medium separately to 37 °C. (ii) Wash ESdMs once with 10 ml of prewarmed DPBS and aspirate with a glass pipette. (iii) Add 10 ml of N2 medium to the ESdMs and scrape them with a cell scraper. (iv) Transfer cell suspension to a 15-ml tube and centrifuge at 300g for 5 min at RT. (v) Remove the supernatant and resuspend the pellet in 1 ml of N2 medium. (vi) Count cells with Neubauer chamber. (vii) Place eight plate inserts into eight wells of a 24-well plate (two wells for each of the conditions outlined below).

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(viii) Transfer 600 µl of N2 medium into each of the eight wells and add 400 µl of N2 medium containing ~100,000 ESdMs (from Step 64A(v)) into each of the inserts.

(ix) Incubate cells for 1 h at 37 °C in 5% CO2. (x) Add different concentrations of CX3CL1 into the wells:

Wells 1–2 0 ng ml − 1 CX3CL1

Wells 3–4 10 ng ml − 1 CX3CL1 (added to the well)

Wells 5–6 20 ng ml − 1 CX3CL1 (added to the well)

Wells 7–8 Control, add 20 ng ml − 1 CX3CL1 into the well and the insert

(xi) Incubate 3–4.5 h (depending on the passage of the cells, incubate longer with higher passages) at 37 °C in 5% CO2. (xii) Remove non-migrated ESdMs from the inserts using cotton buds.(xiii) Count the number of ESdMs that have migrated to the back of the membrane using a Neubauer chamber.(xiv) Calculate the total number of migrated cells, calculate the mean value and normalize to 0 ng ml − 1 CX3CL1

(undirected migration).(b) phagocytosis assay ● tIMInG 30 min on day 1; 10 min on day 2; 3 h on day 3 (i) Day 1: Prewarm DPBS and N2 medium separately to 37 °C. (ii) Wash ESdMs once with 10 ml of DPBS and aspirate with a glass pipette. (iii) Add 10 ml of N2 medium to the ESdMs and scrape cells with a cell scraper. (iv) Transfer cell suspension in a 15-ml tube and centrifuge at 300g for 5 min at RT. (v) Remove the supernatant and resuspend the pellet in 10 ml of N2 medium. (vi) Count the cells with a Neubauer chamber. (vii) Transfer 2 ml of N2 medium containing ~100,000 ESdMs to each well of a 6-well plate.(viii) Incubate cells for 1 day at 37 °C in 5% CO2. (ix) Day 2: Add 500 ng ml–1 LPS to cells and incubate for 24 h at 37 °C in 5% CO2. use one well as untreated control. LPS is

added to stimulate the ESdMs. (x) Day 3: Add 10 µl of microsphere beads to each well and incubate at 37 °C in 5% CO2 for 1.5 h. (xi) Prewarm DPBS, trypsin and N2 medium Separately to 37 °C. (xii) Aspirate old medium from the dishes in Step 64B(ix) with a glass pipette, wash several times with DPBS, then

aspirate DPBS. crItIcal step Make sure to wash cells thoroughly to remove any beads attached to the surface of cells.(xiii) Add 1 ml of trypsin and incubate for 3 min at 37 °C in 5% CO2. (xiv) Transfer cells to 5 ml of N2 medium in a 15-ml tube and centrifuge at 300g for 5 min at RT. (xv) Remove the supernatant with a glass pipette and resuspend the pellet in 500 µl of DPBS.(xvi) Analyze cells by flow cytometry. unstimulated cells should be used as a control and treated like the LPS-stimulated cells. We

recommend measuring at least 10,000 cells for each sample at a speed of 500–900 cells per second. use a laser suited for the measurement of excitation of Fluoresbrite polychromatic red microspheres. On the basis of the fluorescence intensity of the cells, calculate the amount of cells that phagocytosed two or more microsphere beads. When interpreting data, keep in mind that the risk of unspecific binding of beads to the ESdMs cannot be completely precluded. ? troublesHootInG

(c) Flow cytometry ● tIMInG: 2 h 30 min, depending on sample number (i) Collect ESdMs using a cell scraper and calculate ~1,000,000 cells for each sample using a Neubauer chamber. Always

use non-stained cells as a control. Primary antibodies used should be chosen in regard to microglia surface markers or ES cell markers that determine the microglial identity of the ESdMs.

(ii) Centrifuge cells at 300g for 3 min at RT. (iii) Incubate cells for 10 min on ice with Fc-receptor blockade by a rat monoclonal antibody against CD16/32

(5 µg ml–1 in DPBS). Do not incubate samples stained with non-biotinylated antibodies with Fc-receptor blockade, as this will lead to a cross-reaction of the antibodies. Perform matching isotype-control staining for each antibody.

(iv) Split ~1,000,000 cells in 500 µl DPBS into 1.5-ml sample tubes; one tube is needed for each primary antibody. (v) Add the desired primary antibody (see table 1) and incubate for 60 min on ice. (vi) Wash three times with DPBS (repeat washing procedure three times: aspirate with glass pipette, resuspend in 500 µl of

DPBS, centrifuge at 300g for 3 min at RT). (vii) Add appropriate secondary antibody in 500 µl of DPBS and incubate for 30 min on ice and in darkness.(viii) In darkness, wash three times with DPBS (see Step 64C(vi)). (ix) Dilute cells in 500 µl of DPBS and pipette cell solution through a 40-µm cell strainer directly before analysis with

cytometer. For cytometer settings, see Step 64B(xvi).

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(D) qrt-pcr for cytokine profile analysis ● tIMInG 6 h (i) Wash ESdMs once with 10 ml of DPBS and aspirate with a glass pipette. (ii) Isolate RNA using for instance the RNeasy kit (Qiagen) according to the manufacturer’s instructions. (iii) Perform a reverse transcription PCR to generate cDNA (using, for example, the SuperScript III First-Strand Synthesis

System for RT-PCR from Invitrogen). (iv) Dilute cDNA to 200 ng µl–1. (v) Prepare the following qRT-PCR master mix for each sample and pipette, in duplicate, into individual wells of a

96-well plate; avoid making bubbles. Include a non-template control (12.5 µl of SYBR Green mix, 1 µl of each primer and 10.5 µl of water).

component amount per reaction Final

SYBR Green mix (2×) 12.5 µl 1×

cDNA (200 ng µl − 1) 3 µl 600 ng

Primer (10 pmol µl − 1) 1 µl each 0.4 pmol µl − 1

Water up to 25 µl

(vi) Tape the plate with an adhesive cover and centrifuge for 1 min at 300g. (vii) use the following thermal cycler parameters (Eppendorf epgradient S Mastercycler)

cycle number Denature anneal extend

1 95 °C for 10 min — —

2–40 95 °C for 10 s 60 °C for 30 s 72 °C for 30 s

(viii) To assess whether a specific product was obtained, perform dissociation curve analysis:

cycle number temperature rate

1 95 °C for 15 s —

2 60 °C for 15 s —

3 95 °C for 15 s —

4 60 °C to 95 °C Ramp up over 20 min

(ix) Analyze obtained data via ∆CT method. Subtract the average cycle threshold (CT) of the control gene (Gm6316) from the average CT of the gene of interest, which results in the value for ∆CT. The relative gene transcription is the result of 2 − ∆CT.

● tIMInGSteps 1–8, MEF culture: 60 minSteps 9–14, ES cell thawing: 30 minStep 15, ES cell expansion: 10 min per day for 2–3 dSteps 16–22, ES cell passaging: 30 minSteps 23 and 24, Induction of embryoid body formation: 40 min on day 0; incubate for 1 dSteps 25–36, Formation of embryoid bodies: 30 min on day 1 (Steps 25–31) and 40 min on day 2 (Steps 32–36)Steps 37–49, Neural differentiation: 30 min on day 5 (Steps 37–40); 10 min on days 7, 9, 11 and 13 (Steps 41–44) and 10 min daily on days 14–21 (Steps 46–49)Steps 50–56, Microglial differentiation: 10 min on day 21 and every other day thereafterSteps 57–63, Culture of ESdMs: 10 min every other day, 30 min every fourth dayStep 64 (option A), Migration assay: 7–8.5 hStep 64 (option B), Phagocytosis assay: 30 min on day 1 (steps i–viii); 10 min on day 2 (step ix) and 3 h on day 3 (steps x–xvi)Step 64 (option C), Flow cytometry: 2 h 30 min, depending on sample numberStep 64 (option D), qRT-PCR for cytokine profile analysis: 6 hbox 1, MEF culture preparation: 30 dbox 2, Immunocytochemistry: (beginning on day 13 for nestin and day 30 for Iba1, CD45, β-tubulin III and GFAP) 50 min on day 1 (steps 1–5); 1 h 20 min–2 h 50 min on day 2 (steps 6–12)

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? troublesHootInGTroubleshooting advice can be found in table 5.

antIcIpateD resultsusing this protocol, a homogeneous culture of ESdMs is produced after 60 d at the latest. This method is reliable and relatively easy to perform, but particular attention must be paid to the timing of the different steps and the cycles of medium changing and splitting of cells.

Microscopic analysisDuring the differentiation protocol (Steps 15–56), cells should be monitored on a daily basis, especially with regard to mor-phological changes. During expansion of ES cells (Fig. 1), cells should grow in solitary colonies without touching the neigh-boring colonies (Fig. 1a). After the first step, embryoid bod-ies should be formed. They can be recognized as round cell clusters floating in the medium (Fig. 1b). After 3 d, they should be big enough to be seen with naked eye. After plat-ing the embryoid bodies, a variety of cell types will appear, e.g., contracting heart muscle cells might be seen using the microscope around day 12 (data not shown). During Step 45 of the protocol, neural precursor cells are selected in ITSFn medium and can be stained for nestin (Fig. 2a). During the neural differentiation step, neuronal, glial and microglial cells can be detected (Fig. 1c). Around 10 d after growth factor withdrawal (day 30), cultures should contain different

table 5 | Troubleshooting table.

step problem possible reason possible solution

5–63 Contamination of dishes (bacterial, fungi, mycoplasma)

Improper handling (note: medium in these steps does not contain antibiotics)

Work carefully, under sterile conditions; prepare new medium

8 MEF cells do not attach or cell layer is not confluent

Improper gelatin coating; MEF cells of poor quality

Recheck gelatin solution; prepare fresh MEF cells

15 ES cells differentiate ES cells are too dense (>60% confluence)

Split ES cells earlier

36 Embryoid bodies attach to dish Tissue culture dish used Make sure to use noncoated Petri dishes for this step

56 ESdMs proliferate poorly after picking

Picking causes mechanical stress for cells Add 20 ng ml − 1 GM-CSF to culture for several days and do not split until the dish is confluent

57 ESdMs change morphology to activated state

Possible effect of mechanical stress due to splitting too often

Add 20 ng ml − 1 GM-CSF for several days to culture, split less and more gently using a cell scraper; change the medium more often

ESdMs are in poor condition after thawing

Cells were kept in freezing medium for too long or thawed too slow

Immediately freeze cells after transferring them into freezing medium, thaw cells very quickly using a water bath and wash them immediately with N2 medium

64B(xvi) High unspecific fluorescence (beads attached to cell surface)

ESdMs were not washed thoroughly Wash samples more often with DPBS

aES-cell culture

Day 0: Split ES cells to gelatine-coated culture dish in ES medium

b

c

d

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Embryoid body formation

Day 1: Transfer ES cells to Petri dish in DIFF mediumDay 2: Transfer embryoid bodies to new Petri dish

Neural differentiation

Microglial differentiation

ESdM culture

Day 21: Switch to N2 medium with laminin

Day 51 or later: Immunostaining for lba1/CD68 (Box 2)Day 50: Pick ESdM colonies mechanically

Day 51 onwards: Change N2 medium every second day, split on a confluence of 80%

Day 60 onwards: Characterization of ESdMs (flow cytometry, RT-PCR, migration, phagocytosis; Step 63)

Day 30: Immunostainig for lba1, CD45, β-tubulin III, and GFAP (Box 2)

Day 26: Switch to N2 medium

Day 14: Switch to N2 medium with FGF2 and lamininDay 13: Immunostaining for nestin (Box 2)Day 7: Switch to ITSFn mediumDay 5: Plate embryoid bodies on dish with gelatin

Figure 1 | Flow chart of the protocol (Steps 15–57) with bright-field microscopic images. (a) Culture of mouse ES cells on a MEF layer. Scale bar, 200 µm. (b) Embryoid body formation on Petri dishes. Scale bar, 100 µm. (c) Neural differentiation of progenitor cells derived from the embryoid bodies. Scale bar, 100 µm. (d) Colony of microglial precursor cells derived from neural progenitor cells. Scale bar, 100 µm. (e) Culture of ESdMs. Scale bar, 100 µm. All images were collected with an Axiovert 40 CFL microscope (Zeiss) equipped with a Powershot G9 camera (Canon).

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cells expressing β-tubulin III, GFAP, CD45 or Iba1 (Fig. 2b and c). The first microglial cells appear as round, shiny cells within the culture and start to proliferate and build ESdM ‘nests’ (Fig. 1d). After 50 d, cells are positive for Iba1 and express CD68 (Fig. 3a), indicating that the protocol has resulted in a relatively pure ESdM culture (Fig. 1e). The obtained ESdM line should be analyzed by a combination of additional approaches to define its properties, as described below.

cell surface protein expression of esdMs determined by flow cytometryAs a variety of antibodies for surface markers are available, ESdMs can additionally be analyzed by flow cytometry (Step 64, option C). ESdMs show the expression of typical microglial markers. The antibodies used for flow cytometry are listed in tables 3 and 4.

ESdM lines show a high expression of the microglial surface molecules CD11b, CD11c, CD29, CD36, CD45, CD80, CD86 and CD115, whereas stem cell population markers such as CD34 or CD117 are undetectable or only weakly expressed (see Fig. 3b). This lack of stem cell population markers indicates that the ESdMs are fully committed to differentiation.

transcript levels of esdMs determined by qrt-pcrESdMs can be stimulated in vitro by LPS (500 ng ml − 1) or IFN-γ (500 u ml–1), which typically leads to an increase in phagocytosis and the production of proinflammatory mediators such as

a DAPI Nestin Merge

c DAPI

CD45

GFAP

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b

Iba1

DAPI

Merge

β-Tubulin III

Figure 2 | Immunocytochemistry of cells during neural and microglial differentiation (Steps 37–56). (a) Neural precursor cells at day 13 of the protocol. Cells were immunostained against nestin and nuclei were counterlabeled with DAPI. Scale bar, 20 µm. (b) Mixed neural culture. Cells were immunostained with antibodies against Iba1 and β-tubulin III. Microglia-like cells were detected as Iba1-positive cells within the neuronal network labeled by the β-tubulin III–specific antibody. Scale bar, 50 µm. (c) Co-immunostaining of GFAP and CD45 at day 30 of the protocol. No colocalization of CD45-positive microglial precursors and GFAP-positive astrocytes was observed. Scale bar, 50 µm. All images were collected with an Axioskop 2 fluorescence microscope (Zeiss) equipped with a camera.

100

Iba1

CD11b CD11c CD29 CD34

CD86CD80CD45CD36

CD115 CD117

CD68 Mergea

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Figure 3 | Analysis of ESdM surface markers. (a) Cells immunostained with an antibody against Iba1 were also positive for the marker CD68. Scale bar, 50 µm. Images were collected with a Fluoview FV 1000 confocal microscope (Olympus). (b) Flow cytometric analysis showed that typical microglial surface markers such as CD11b, CD11c, CD29, CD36, CD45, CD80, CD86 and CD115 were expressed by ESdMs. Expression of stem cell markers CD34 and CD117 was very low or almost undetectable in ESdMs. Analyzed samples are shown in red; isotype controls are shown in blue.

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tumor necrosis factor-α and nitric oxide synthase 2. Cytokine levels can be analyzed using real-time PCR (Step 64, option D). Possible primers for the detection of relevant cytokines are listed in table 6. Both treatments should result in an at least twofold increase of mRNA transcripts of nitric oxide synthase 2 and tumor necrosis factor-α (see Fig. 4a).

cell migration assay of esdMsTo provide an insight into the functional properties of ESdMs, a variety of experiments are available. In the CNS, the fractalkine CX3CL1 is one of the most prominent chemokines expressed by neurons. The corresponding receptor, CX3CR1, is expressed by microglia, leading to a chemotaxis reaction of the microglia with the fractalkine. Therefore, one would expect a similar migration rate of ESdMs on exposure to CX3CL1. A suitable experiment to verify the migration rate would be a cell migration assay including a CX3CL1 concentration gradient (Step 64, option A). We have shown that the chemokine-directed migration of ESdMs increased nearly twofold up to 196 ± 25.85% in comparison to non-directed migration for the highest concentration of CX3CL1 (Fig. 4b).

ESdMs should migrate in a chemokine-directed and concentration-dependent manner, and the migration rate at 20 ng ml − 1 CX3CL1 should be at least 50% higher than in the control. A positive result of this experiment suggests that the ESdMs show directed migration toward CX3CL1, similar to what would be expected in primary microglia.

phagocytosis assay of esdMsPhagocytic capability can be measured easily by evaluating the uptake of labeled microsphere beads by flow cytometry (Step 64, option B). On treatment with 500 ng ml − 1 of LPS, ESdMs react with an increase in microsphere uptake compared with untreated control cells; this indicates an activated state of the ESdMs (Fig. 4c). unfortunately, flow cytometry can-not distinguish between cells that have ingested beads and cells that have beads attached to the cell surface. Therefore, only cells with a higher fluorescence signal than that from one bead should be taken into account using this method. A threshold for unspecific fluorescence from beads which were not taken up by ESdMs could be set using cytochalasin-D-treated ESdMs as a control, as cytochalasin-D blocks phagocytosis. A culture of functional cells should contain at least 10% phagocytically active microglia under unstimulated conditions. On stimulation with LPS, the percentage of ESdMs showing phagocytosing capability has increased by ~50%.

table 6 | List of oligonucleotide sequences used for qRT-PCR.

name Forward sequence reverse sequence

Gm6316 5′-ACAACTTTGGCATT GTGGAA-3′

5′-GATGCAGGGATGATG TTCTG-3′

Nos2 5′-AAGCCCCGCTACT ACTCCAT-3′

5′-GCTTCAGGTTCCTGA TCCAA-3′

Tgfb1 5′-CAATTCCTGGCGTT ACCTTG-3′

5′-GCTGAATCGAAAGCCC TGTA-3′

Tnf 5′-TCTTCTCATTCCTG CTTGTGG-3′

5′-AGGGTCTGGGCCA TAGAACT-3′

104 250

200

150

100

50

00 10 20 Control

CX3CL1 (ng ml–1)

LPS

31.4 %20.9 %

3 beads3 beads

104104

Fluorescence intensity

Cel

l cou

nt

00

300300

1,2001,200

Untreated

900900

600600

103103 102102 101101 100100

2 beads

2 beads

1 bead1 bead

103

a b

c

102

101

100

10–1

NOS2

Rel

ativ

e ge

ne tr

ansc

riptio

n

Rel

ativ

e m

igra

tion

(%)

TNFα

Control IFNγLPS

TGFβ

Figure 4 | Functional analysis of ESdMs. (a) Relative cytokine gene transcription of ESdMs after treatment with IFN-γ (500 u ml–1) or LPS (500 ng ml–1) for 24 h. Treatment with IFN-γ resulted in an increase in Nos2 (~1,000 fold) and Tnf (TNFα, ~20 fold) gene transcription. Gene transcripts of Tgfb1 (TGF) were almost unaffected by the treatment. Error bars, mean ± s.e.m. Mean value of four experiments. (b) Migration assay of ESdMs toward the chemokine CX3CL1. Number of migrating cells increased in a dose-dependent manner on a CX3CL1 gradient. Chemoattraction by 20 ng ml–1 CX3CL1 led to an increase of almost 100% of relative migration. As a control (Control), 20 ng ml–1 CX3CL1 was added to the ESdMs without gradient. No increased migration was observed with non-directed stimulation of CX3CL1. Error bars: mean ± s.e.m. Mean value of three independent experiments. (c) Phagocytosis assay. uptake of microsphere beads was analyzed by flow cytometry. The number of beads taken up was determined by differences in fluorescence intensity (see arrows in Fig. 3c). uptake of two or more red fluorescently labeled microsphere beads by a single ESdM was regarded as a phagocytically active cell. upon stimulation with 500 ng ml–1 LPS, the percentage of ESdMs showing uptake of two or more microspheres was increased from 20.9% to 31.4%. Please note that cells that have not phagocytosed any beads are not shown in the fluorescence intensity histogram due to the flow cytometer settings.

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acknowleDGMents The Neural Regeneration Group at the university of Bonn Life & Brain Center is supported by the Hertie Foundation, the Walter und Ilse Rose Foundation, the Deutsche Forschungsgemeinschaft (FOR1336, KFO177, SFB704) and the Eu (LSHM-CT-2005-018637). We thank J. Schumacher and R. Hass for excellent technical support of cultures and molecular biology.

autHor contrIbutIons C.B. and H.N. conceived and designed the experiments. C.B. performed generation of ESdMs and did most of the characterization of the cells by immunocytochemistry, flow cytometry and qRT-PCR. K.R. carried out the phagocytosis assay and parts of the characterization by flow cytometry. B.L. defined the purity of the cell population and analyzed the migratory capacity of the cells. C.B., K.R., B.L. and H.N. wrote the paper. I.N. participated in the establishment of the generation protocol of ESdMs from ES cells and corrected the paper.

coMpetInG FInancIal Interests The authors declare no competing financial interests.

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