LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

All protocol steps adhere to the guidelines set forth by the NIH's human research ethics committee. Stem cell work and patient sample collection were approved by the Combined NeuroScience Institutional Review Board (CNS IRB) under the Office of Human Research Protection (OHRP), NIH, as per 45 CFR 46 guidelines of the U.S. Government. Patient samples were collected using CNS IRB-approved consent form in accordance with the criteria set by the Declaration of Helsinki under the protocol number NCT01432847 (https://clinicaltrials.gov/ct2/show/NCT01432847?cond=NCT01432847&draw=2&rank=1).

1. iRPE generation

1. Generate iRPE from patient blood-derived iPSC following the published protocol by Sharma et al., 2022¹¹ (Figure 2 and Figure 3).

Figure 2: Schematic of iRPE differentiation and maturation. To generate iRPE, an established differentiation protocol was followed, and the cells were allowed to mature for 5 weeks. The resulting cell culture acts as an in vitro model that can be manipulated with various treatments to mimic RPE dysfunction in diseases like AMD and Stargardt disease. Please click here to view a larger version of this figure.

Figure 3: Representative images of successful and unsuccessful RPE differentiation and maturation. Two brightfield images at 10x magnification of TJP1 RPE are shown at Day 42 of the iRPE protocol. (A) Successful differentiation and maturation will show confluent RPE with pigmentation and polygonal morphology. (B) Unsuccessful differentiation and maturation will show clusters of dying cells, as shown here. Please click here to view a larger version of this figure.

2. RPE maintenance media (RPE-MM) preparation

1. Thaw the N2 supplement at 4 °C overnight. Thaw all other reagents at room temperature (RT).
2. Under sterile conditions, add the reagents listed in Table 1 at the listed dilution factors, as per the protocol established by Sharma et al., 2022¹¹.
3. Mix the media well and filter it using a 0.22 µm filtration unit.
   ​NOTE: Media is suitable for use within 2 weeks if stored at 4 °C.

3. 96-well plate seeding

1. Thaw an aliquot of vitronectin at RT for 3-5 min or until the ice is completely melted.
2. Dilute the vitronectin with 1x Dulbecco's phosphate-buffered saline (DPBS) to obtain the desired working solution using a 1:200 dilution (vitronectin: DPBS). For a 96-well plate, coat each well with 200 µL of the working solution.
3. Combine thawed ROCK inhibitor (Y-27632 dihydrochloride) with the RPE-MM at a 1:1000 dilution to achieve a final concentration of 10 µM. This is the plating media for RPE cells.
4. Thaw the iRPE vial using an automated cell thawing system and transfer the iRPE cell suspension to a 50 mL tube.
5. Dilute the cell suspension with the plating media at a 1:10 dilution. Centrifuge the tube at 400 x g for 5 min.
6. Carefully aspirate the supernatant and resuspend the cells in 10 mL of the plating media.
7. Mix 400 µL of the plating media with 100 µL of the resuspended cell solution for cell counting. Use this aliquot to determine the viable cell concentration of the cell suspension using a cell viability counter.
8. Dilute the cell suspension with the plating media to a final concentration of 60,000 cells/mL.
9. Completely aspirate the vitronectin coating solution from the 96-well plate and dispense 200 µL of the cell suspension into each well. There will be roughly 12,000 cells/well or ~200 cells/mm².
10. Incubate the seeded cell plates for 48 h at 37 °C and 5% CO₂. After 48 h, change the media to RPE-MM without ROCK inhibitor supplement. Change the media every 2-3 days during the 5-week maturation period.

4. In vitro disease models

1. Complement competent human serum (CC-HS) treatment
   1. Thaw human complement competent serum at 4°C overnight.
   2. Prepare CC-HS and complement incompetent human serum (CI-HS) media.
      1. To prepare 5% CC-HS media, mix the thawed complement competent human serum with RPE-MM at a 1:20 dilution. Filter the solution through a 0.22 µm media filter before use.
      2. To prepare 5% complement incompetent human serum (CI-HS) media, first heat inactivate the CC-HS in a 57 °C water bath for 30 min and then mix with the culture media at a 1:20 dilution. Filter the solution through a 0.22 µm media filter before use.
   3. Serum treat the cells with 200 µL of either 5% CC-HS or 5% CI-HS media for a total incubation time of 48 h, refreshing the media after 24 h.
   4. Wash the cells with 1x DPBS and fix them with 4% paraformaldehyde for 20 min at RT. Wash once more with 1x DPBS and store the samples at 4 °C, immersed in 200 µL of DPBS.
   5. OPTIONAL: If desired, lyse the cells from the plate to show only sub-RPE lipid deposition.
      1. To lyse the cells and leave only lipid deposits, remove the media and add 200 µL of deionized water to each well.
      2. Incubate for 10-15 min, pipette up and down until cells are removed. Wash once more with 200 µL of deionized water and immediately fix the cells with 4% paraformaldehyde.
      3. Confirm the cell removal efficacy with nuclear staining using Hoechst. Add Hoechst at 1:2000 dilution to a 1x DPBS solution containing 1% bovine serum albumin (BSA), 0.5% Tween 20, and 0.5% Triton-X 100. Incubate at RT for 1 h in the dark. Subsequently, wash with 1x DPBS.
2. Photoreceptor outer segment (POS) treatment on iRPE
   1. POS preparation
      1. Remove the POS pellet tube from -80 °C storage and thaw overnight at 4 °C in a covered ice bucket.
      2. Prepare POS wash buffer by mixing 10 g of sucrose in 40 mL of double-deionized H₂O (ddH₂O).
      3. Heat mixture at 40-50 °C while gently stirring for 15 min. Add 840 mg of sodium bicarbonate to the mixture and stir while heating for 10 min.
      4. Adjust the total volume of the POS wash buffer to 100 mL with ddH₂O and adjust the pH of the solution to 8.3 with 1 N HCl or 1 N NaOH, as needed. Filter the wash solution using a 0.22 µm filter.
         NOTE: The protocol can be paused here; the POS wash buffer can be stored at 4 °C overnight.
      5. Once thawed, suspend the pellet in 15 mL of POS wash buffer. Be gentle during pellet suspensions to ensure POS integrity. Centrifuge the POS suspension at 600 x g at 4 °C for 20 min and then aspirate the supernatant.
      6. Resuspend the POS pellet in 10 mL of the POS wash buffer.
      7. Remove a 100 µL aliquot of the POS + POS wash buffer (POS solution) and dilute in 400 µL of 1x DPBS. Spread 50 µL of the diluted POS solution on a blood agar plate and an agarose plate to check for bacterial and fungal contaminants. Prepare positive controls for each and incubate all plates for 48 h at 37 °C.
      8. Perform a qPCR assay by adding 1 µL of POS solution to the detection well to test for mycoplasma. To amplify DNA fragments, perform 40 cycles of denaturation (95°C, 15 s), and annealing and elongation (60°C, 1 min). The forward and reverse primers for detecting mycoplasma in the POS sample are as follows:
         Forward primer: GGA TTA GAT ACC CTG GTA GTC CAC G
         ​Reverse primer: CGT CAA TTC CTT TAA GTT TCA CTC TTG GC
      9. Measure POS concentration using a cell analyzer and aliquot according to need. For one well of 96 well plates with RPE cells, 3 x 10⁶ POS is sufficient. The desired ratio is 10 POS/RPE cell. Store the aliquots at 80 °C for future use.
   2. Addition of POS to cells
      1. Thaw vials of POS in an ice bath.
      2. Mix the calculated amount of prepared POS with RPE-MM and treat the cells with POS once daily for 7 days.
         ​NOTE: Prepare POS solution fresh daily.
      3. Wash the cells with 1x DPBS and then fix them with 4% paraformaldehyde for 20 min at RT. Wash with DPBS once more and store samples at 4 °C, immersed in 200 µL of DPBS.

5. Staining for sub-RPE deposits

1. Nile red staining protocol
   1. After PFA fixation, wash the samples 3 times with 1x DPBS.
      ​NOTE: If not used immediately, the protocol can be paused here, but samples must be stored in a 1x DPBS + 0.02% sodium azide solution at 4 °C.
   2. To prepare the Nile Red stock solution, dissolve Nile Red powder in acetone at 3 mg/mL concentration. Incubate for 15 min at RT with periodic mixing. Filter the solutions with a 0.22 µm filter once or twice depending on the level of precipitate remaining in the solution.
      NOTE: Protect the stock solution from light.
   3. To prepare the working solution, dilute the stock solution at a ratio of 1:500 in 1x DPBS. Add 200 µL of the working solution to the sample for 30 min at RT on a shaker and protect it from light.
   4. Wash 3 times with 1x PBS and store the samples at 4 °C, immersed in 200 µL of DPBS.
      NOTE: If conducting an experiment on transwells instead of a 96-well plate, samples can be mounted on a slide with mounting media, covered with a glass coverslip, and sealed with transparent nail polish. Care should be taken to mount the sample with the cells facing up.
2. BODIPY staining protocol
   1. For stock solution, dissolve BODIPY in anhydrous dimethyl sulfoxide (DMSO) to attain a stock concentration of 3.8 mM.
   2. For PFA fixed samples, dilute BODIPY stock at 1:300 in 1x DPBS. Add 200 µL to cells and incubate overnight on a rocker at RT.
   3. Wash 3 times with 1x DPBS and store the samples at 4 °C, immersed in 200 µL of DPBS.
      NOTE: If conducting an experiment on transwells instead of a 96-well plate, samples can be mounted on a slide with mounting media, covered with a glass coverslip, and sealed with transparent nail polish. Care should be taken to mount the sample with the cells facing up.
3. APOE immunostaining protocol
   1. Combine 1x DPBS with 1% bovine serum albumin (BSA), 0.5% Tween 20, and 0.5% Triton-X 100 to create a buffer solution.
   2. For PFA fixed samples, block and permeabilize the sample in 200 µL of the buffer solution for 1 h at RT.
   3. Add APOE primary antibody diluted at 1:100 in the buffer solution and incubate overnight at RT.
   4. The following day, wash the samples 3 times with 1x DPBS.
   5. Add a secondary antibody at 1:1000 dilution in the buffer solution and add 200 µL of the solution to the cells for 1 h at RT.
   6. Wash 3 times with 1x DPBS and store samples at 4 °C, immersed in 200 µL of DPBS.
      ​NOTE: If conducting an experiment on transwells instead of a 96-well plate, samples can be mounted on a slide with mounting media (Fluoromount), covered with a glass coverslip, and sealed with transparent nail polish. Care should be taken to mount the sample with the cells facing up.

6. Image automation and processing

1. Automated image scan
   NOTE: Zeiss LSM 800 inverted confocal scanning microscope and ZEN 3.2 (blue edition) software were used in this study. Ensure the 96-well plate is warmed to RT for a minimum of 60 min before imaging to avoid focal plane drift during the scan due to a change in the refractive index of the medium with temperature change.
   1. Using a confocal microscope and a 40x objective, create a scan profile with the appropriate fluorescent channels for the lipid marker used and any additional antibodies.
   2. Use the Tiles checkbox to set up image automation. To calibrate the 96-well plate, ensure the correct sample carrier measurements are entered and selected. Then, click the Calibrate button to calibrate the plate according to the instructions, which requires using the 10x objective.
   3. Choose the Advanced Setup view to select appropriate wells and add 3 different imaging points near the center of the well using the Positions function. This can be done manually under the Position subtab or randomly using the Position Setup tab and selecting Setup by Carrier. Repeat for all the wells of the same staining.
   4. For optimal focusing and Z-stack positioning during automation, go to the Focus Strategy tab to select Use Focus Surface/Z values Defined by Tiles Setup. Alternative methods can use other focus strategies, but it is recommended to use this setting for the most consistent results.
   5. Under the Tiles tab, click Verify Positions and manually set the central Z plane for each position. The settings in the Options subtab will order the acquisition of images, so check this before starting to image. To acquire images in the order the positions were selected, deselect the Tile Regions/Positions and Carrier Wells/Container checkboxes. Select Split Scenes into Separate Files for ease of image processing.
   6. Ensure the Z-Stack tab is set to Center, a range is inputted to the user's preferences, and the Optimal button is selected to set the slice interval.
   7. After optimizing the Acquisition Mode, Channels, Focus Strategy, Z-Stack, and Tiles tabs, start the experiment.
2. Image processing
   1. Using a batch image processing method, create maximum projections of each Z-stack with the Extended Depth of Focus method.
   2. Using a batch image processing method, export the maximum projection files as 16-bit TIFF images. Set the compression to None and ensure Original Data is checked. The resulting image should be a maximum projection greyscale TIFF of only the fluorescence channel on which the lipid marker is expressed.

7. Segmentation and quantification

NOTE: The LipidUNet program was trained on 40x images from a 96-well plate. It is highly recommended to use images that were obtained using a 40x objective.

1. Install the LipidUNet software. LipidUNet can be downloaded from the following GitHub repository:  https://github.com/RPEGoogleMap/LipidUNet
2. Identify the TIFF images representing either Nile Red, Bodipy, or APOE and move them into a folder named imgs within a directory named either Nile_Red, Bodipy, or APOE, depending on the method being used.
   NOTE: Exact naming conventions must be used for the LipidUNet program to recognize the directories.
3. Open the LipidUNet software (Figure 4).
4. In the Predict tab of the software, select the relevant directory (Nile_Red, Bodipy, or APOE) by clicking on the ellipsis and navigating to the named directory. Confirm that the LipidUNet program has correctly identified the images by checking the Class entry.
5. Select a probability threshold for the algorithm between 0.01 and 0.99. A higher value will eliminate more false positives but might cause more false negatives, and lower values might introduce more false positives while eliminating more false negatives. A value of 0.65 is the default and is recommended.
6. Click Predict.
   ​NOTE: The software will iterate through all the images automatically and create a new folder called predicted_masks in the selected directory.
7. Use a Mask Analysis tool to iterate through the generated masks and provide a quantitative count of the thresholded lipid deposits from the mask images.
8. Analyze the generated counts data to compare treatment conditions.

Figure 4: LipidUNet user interface. The LipidUNet software has different sections to select for the training data directory, where images of lipid deposits have been identified correctly; the model weights directory, which is produced from the training data; and the prediction data directory in which the user will input their images for segmentation. Please click here to view a larger version of this figure.