iPS-derived microglia represent a biologically relevant source of human microglia for in vitro experimentation, and are an important tool to investigate microglia biology in health and disease. We present a simple microglia differentiation protocol that requires minimal use of growth factors and achieves high purity and yield. Also, we demonstrate a fully humanized phagocytosis assay.
iPSC-derived microglia-like cells are very sensitive to media evaporation. You should avoid using the wells in the corner of the cell culture plate, and fill all empty wells with water to improve survival. Once Induced Pluripotent Stem Cells, or iPSCs, have reached 80%confluency, dissociate the colonies by washing with one milliliter of DPBS, and adding one milliliter of a dissociation reagent for two minutes at 37 degrees Celsius.
Dislodge the colonies using a cell lifter by scraping multiple times to create a single cell suspension. Collect the suspension, and transfer them to a 15-milliliter conical tube containing nine milliliters of DPBS. Then centrifuge the cells at 500 times g for one minute, remove the supernatant, and resuspend in one milliliter of the Embryoid Body, or EB, medium.
Take 10 microliters of cells, and dilute one to two with trypan blue. Count the cells on a hemocytometer, and based on the cell count, dilute the cell stock to a final dilution of 10, 000 cells per 100 microliters. For plating cells, add 100 microliters of the diluted cells per well into a low adherence round-bottom 96-well plate.
Centrifuge the plate at 125 times g for three minutes, and incubate at 37 degrees Celsius and 5%carbon dioxide for four days. On day two, using a multi-channel pipette, replace the old half-EB medium with a fresh medium. To generate Primitive Macrophage Precursors, or PMPs, coat the wells of a six-well plate by adding one milliliter of ice cold Matrigel coating solution.
Then incubate the plate at 37 degrees Celsius and 5%carbon dioxide for two hours, or overnight. On day four of EB differentiation, using a one-milliliter pipette tip, transfer the EBs to the Matrigel-coated wells. Pipette up and down to dislodge the EBs from the well.
Then hold the plate tilted to allow the EBs to settle down at the edge of the well. Once all the EBs have settled down, gently pipette and replace the old medium, while keeping the EB at the edge of the well with three milliliters of freshly prepared PMP complete medium. Evenly distribute the cells in the wells by manually shuffling the plate side to side and back to front.
Then incubate the plate for seven days to allow the EBs to attach to the bottom of the well. After seven days, inspect the EBs under a light field microscope at four times magnification to ensure that they are attached to the bottom of the wells. Perform a half medium change, and on day 21, replace the complete medium with three milliliters of PMP complete medium.
On day 28, look for round cells referred to as PMPs in the supernatant. Then collect the medium containing the PMPs using a 10-milliliter pipette and automatic pipetter without disturbing the EBs. Transfer the PMPs in the medium to a 15-milliliter conical tube.
Centrifuge the collected PMPs at 200 times g for four minutes and aspirate the supernatant before re-suspending them in one to two milliliters of microglia-like cells, or IMG basal medium. Then count the cells on the hemocytometer as described previously. Centrifuge the rest of the cells, and dilute the PMPs to the desired concentration.
Then plate the cells at a density of 10 to the fifth cells per square centimeter on cell culture-treated plates using freshly prepared IMG complete medium. For live cell phagocytosis assay, plate 20 to 30 times 10 to the fourth PMPs into a 96-well plate and 100 microliters of IMG complete medium and follow the differentiation process for 10 days. On the day of the assay, remove 40 microliters of medium per well, and add 10 microliters of the nuclear staining solution.
Incubate the plate for two hours. Thaw the labeled synaptosomes on ice, and gently sonicate using a water sonicator for one minute. Again, immediately place the synaptosomes on ice.
Dilute the labeled synaptosomes in IMG complete medium at one microliter of synaptosomes per 50 microliters of medium. For negative control, prepare 60 micromolar Cytochalasin D in IMG complete medium to inhibit actin polymerization, and thus phagocytosis. Then add 10 microliters of this solution to each well for a final concentration of 10 micromolar, and incubate for 30 minutes.
Remove the plate from the incubator, and incubate at 10 degrees Celsius for 10 minutes. Maintain the plate on ice, and add 50 microliters of medium containing synaptosomes. Centrifuge the plate at 270 times g for three minutes at 10 degrees Celsius, and maintain the plate on ice until imaging acquisition.
Insert the plate into a live cell imaging reader, and select the wells to be analyzed. Then select a 20X objective lens. Next, adjust the focus, Light Emitting Diode, or LED, intensity, integration time, and gain of the bright-field and blue channels.
The synaptosome fluorescence should be negligible at the initial time point. Select the number of individual tiles to be acquired in a montage per well. Acquire 16 tiles at the center of the well, imaging approximately 5%of the total well area.
Set the temperature to 37 degrees Celsius, and the desired time interval for imaging. Next, open the analysis software. Then open the experiment that contains the images, and click on the Data Reduction icon.
In the menu, pick Imaging Stitching under Imaging Processing to create a complete image from the four by four individual tiles in the montage with the parameters described in the text manuscript. Define an intensity threshold using the DAPI and RFP channels for these images. Open an image, and click on Analyze.
Under Analysis, select Cellular Analysis, and under Detection Channel, pick a stitched image either in the DAPI, or RFP channels. Next, go to the Primary Mask and Count tab, and establish a threshold value and object size values that properly select the cell nuclei in the DAPI channel, or the synaptosome signal in the RFP channel. To count the number of nuclei, go to the Data Reduction menu, and under Image Analysis, select Cellular Analysis.
Again, go to the Primary Mask and Count tab, and under Channel, select the DAPI stitched images, and use the parameters described in the text manuscript. Next, go to the Calculated Metrics tab, and select Cell Count. Then to obtain the area of the synaptosome signal, go to the Data Reduction menu, and under Analysis, select Cellular Analysis.
Again, go to the Primary Mask and Count tab, and under Channel, select RFP stitched images, and use the parameters detailed in the text manuscript. Next, go to the Calculated Metrics tab, and select Object Sum Area. In the Data Reduction tab, click on OK to allow the software to analyze all the acquired images.
Once the images are analyzed, export the Object Sum Area and Cell Count values for each time point. Then divide the Object Sum Area by the Cell Count to calculate the normalized area per time point. If comparing multiple treatments, or genotypes, calculate the phagocytosis index using the given equation.
Undifferentiated iPSCs show compact colony morphology with well-defined edges. Dissociated iPSCs formed spherical aggregates termed EBs, and grow in size until day four of differentiation. When the EBs are plated for PMP generation, they attach to the Matrigel-coated plates, and a layer of cells spreads and surrounds the spherical aggregates.
On day 28, round cells with a large cytoplasm to nucleus ratio appear in the suspension. It is strongly recommended to culture iPSCs in Laminin 521-coated plates instead of Matrigel, because of the higher PMP yield in Laminin 521-coated plates. After the exposure of PMPs to IMG medium, cells acquire a microglia-like morphology with a small cytoplasm in the presence of elongated processes.
Further, microglia identity was verified by immunofluorescent staining. Typically, more than 95%of the cells express IB1 and approximately 90%of the cells express P2RY12 and TMEM119. Western blot analysis confirmed that iPSC derived lower motor neurons for human synaptosomes'expressed synaptic markers, synaptophysin and postsynaptic density protein 95.
In control compared to the initial time point, by 10 hours, the red fluorescent signal was robust, as most of the synaptosomes had been engulfed, and were localized to acidic intracellular compartments. In Cytochalasin D-treated IMGs, a strong reduction of the red signal for zero and 10 hours indicated that the detected signal results from phagocytic events. The area of engulfed synaptosomes increased with time until 16 hours.
However, the area of red signal from Cytochalasin D-treated cells did not increase with time. iPS-derived Microglia like cells can be used for various applications, including phagocytosis, and inflammatory response in the study of neurodevelopmental and neurodegenerative diseases.
