Genetics
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Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations
Chapters
Summary August 24th, 2018
Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.
Transcript
This method can help answer key questions in the ion-channel related retinal diseases field about the molecular mechanisms of BEST1 mutations and pathology of bestrophinopathies. The main advantage of this technique is that it circumvents the difficulty of directly obtaining RPE cells from humans. The implications of this technique extend toward the therapy of bestrophinopathies, including Best disease, as the RPE cells can be used for both research purposes and cell replacement therapies.
When the hPSC culture reaches confluency, replace the culture medium with four milliliters of differentiation medium per well of a six-well plate. After two weeks of culture, supplement the differentiation medium with 100 nanograms per milliliter of human activin-A, changing the supplemented media three times a week for another 14 days. On day 29, switch to non-supplemented differentiation medium for another eight to 10 weeks until pigmented RPE cell clusters appear.
To isolate the pigmented RPE cells, first hold individual nine-inch glass Pasteur pipettes horizontally over the flame of a Bunsen burner, about two-thirds down the length of the thin part of the pipette. When the heated glass turns soft, quickly pull the two ends apart to form a microscraper-like tip at the new end of the pipette. Spray the pulled pipettes with 70%ethanol and let them air dry in a cell culture hood.
When the pipettes are ready, wash each well of the differentiation culture with PBS and add one milliliter of 0.05%trypsin supplemented with one unit per microliter of collagenase to each well. After 20 to 30 minutes at 37 degrees Celsius, stop the digestion with two milliliters of 37-degrees Celsius RPE medium per well of a six-well plate and place the plate onto a microscope stage. Using a pulled pipette, gently tap the pigmented RPE clusters.
As soon as the cells detach from the bottom of the well, use a 20-microliter micropipette to transfer dissociated cells into individual wells of a new cell culture plate containing RPE medium. When approximately 5, 000 RPE cells have been added to each well of a 12-well plate, bring the final volume up to two milliliters with fresh RPE medium and culture the isolated hPSC-RPE cells for another six to eight weeks until a monolayer of pigmented and cobblestone-shaped mature RPE cells is observed. 24 to 72 hours before the patch clamp, wash the pigmented monolayer cells with two milliliters of PBS, then, after removing the PBS, add one milliliter of 0.05%trypsin plus one unit per microliter of collagenase to the cells in each well of a 12-well plate for an eight-minute incubation period at 37 degrees Celsius.
When the cells begin to detach, use a one-milliliter micropipette to gently wash the cells from the bottom of the well and pool the cell suspension into a 15-milliliter conical tube. Add one milliliter of 37-degrees Celsius trypsin-collagenase solution to the well. Incubate for another eight minutes at 37 degrees Celsius, then wash any remaining cells from the bottom of the well and combine the cell suspension to the same 15-milliliter conical tube.
Incubate the cells within a 37-degrees Celsius dry bath for eight minutes, then use a one-milliliter micropipette to gently triturate the cells 10 to 15 times. After incubating and triturating the cells two more times, add five milliliters of 37-degrees Celsius RPE medium to the single-cell suspension and pellet the cells by centrifugation. Resuspend the pellet in the appropriate volume for seeding onto pre-coated plates with cover slips at 10 to 20%confluency and perform whole-cell patch clamp according to standard protocols.
After successful isolation, greater than 90%of the cells in the passage zero population should mature to display the signature RPE cell morphology. With the hPSC-RPE-based disease-in-a-dish approach, each patient-specific BEST1 mutation can be comprehensively characterized for its protein expression, membrane trafficking, and ion channel function to obtain critical information for elucidating RPE pathological mechanisms. It should be noted that some BEST1 mutations might affect its protein expression, so other well-established RPE markers should also be checked.
Human pluripotent stem RPE cell differentiation takes three to six months, depending on the human pluripotent stem cell line and mutation type, and isolation of the differentiated human pluripotent cells can be completed in two hours if it is performed properly. While attempting to split the cells for the patch clamp, it is important to remember to triturate the cells very gently to avoid excess cell death. Following this procedure, other methods like 3D RPE cell culture can be performed to answer additional questions about how ion transport on the apical and basal membranes of RPE cells is affected by BEST1 mutations.
After its development, this technique paved the way for the researchers in the field of retinal degenerative diseases to explore the physiological phenotypes and pathological mechanisms of patient-derived RPE cells. After watching this video, you should have a good understanding of how to differentiate pluripotent stem cells into retinal pigment epithelium cells and to prepare the RPE cells for whole-cell patch clamp.
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