This protocol describes a simple and efficient method of differentiating human IPCs into complex three-dimensional neuro retinal organoids for research and regenerative applications. This technique involves both adherent and suspension cultures, which allows selective picking and enrichment of both retinal organoids and RP cells. It can offer a viable and regular supply of cell sources for developing cell-based therapies for retinal degenerative diseases.
Such stem cell-derived retinal organoids and RPE cells are useful as in vitro models to study eye development and inherited retinal diseases. This method can be easily replicated by research labs that are already familiar with handling human IPSC cultures. Visual demonstration greatly supports the identification of eye fields and the isolation of neuro retinal cups based on their spatial positioning and morphological features.
To begin, aspirate the spent medium from 70 to 80%confluent human IPSC cultures in six-well plates. Add PBS to the wells, swirl them and aspirate out the wash buffer. Then add one milliliter of cell dissociation solution or CDS to each well and incubate the plate at 37 degrees Celsius for five to seven minutes until the cells round up.
Aspirate the CDS and collect the cell suspension into a 15 milliliter centrifuge tube. Spin the tube at 1, 000 RPM for four minutes at room temperature. Discard the supernatant and resuspend the cell pellet in 1.2 milliliters of Essential 8 medium.
Dispense 200 microliters of the cell suspension into each well of a matrix-coated six-well plate containing 1.5 milliliters of IPSC culture medium and 10 micromolar Y27632. After 12 to 24 hours, remove the spent medium and add pre-warmed complete ascension medium without Y27632 to maintain the cultures. Change the culture medium every 24 hours until the cultures reach 70 to 80%confluence.
On day zero, aspirate out the human IPSC maintenance medium and add differentiation induction medium containing one nanogram per milliliter BFGF and one nanogram per milliliter Noggin to the six-well plate. Incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator. On day one, aspirate out the spent medium and add differentiation induction medium containing one nanogram per milliliter BFGF and 10 nanograms per milliliter Noggin.
Incubate the cells again at 37 degrees Celsius in a 5%carbon dioxide incubator. On day two and three, remove the spent medium and add differentiation induction medium containing 10 nanograms per milliliter Noggin. After adding two milliliters per well medium to a six-well plate, incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator and change the medium every 24 hours.
On day four, remove the spent medium and add the retinal differentiation medium or RDM. Follow the same procedure to add two milliliters per well medium to a six-well plate and to incubate the cultures. Change the medium every day.
At around day 14 and 18, observe the cultures under a microscope at 10X magnification for the emergence of neural rosette-like domains consisting of early eye field progenitors. At around day 21 and 28, observe the cultures under a microscope at 4X and 10X magnification to observe the emergence of self-organized distinct EFPs with a central island of circular 3D neuro retinal structures surrounded by contiguous outgrowths of neuro epithelium and ocular surface epithelium. Take a sterile Pasteur pipette and hold the base in one hand and the capillary tip in the other.
Flame sterilize and heat the region near the middle of the capillary tip with rotational movements until the glass becomes pliable. Then move away from the flame and swiftly pull the capillary tip outward to create a fine capillary tip with a closed lumen. Hold the fine tip horizontally in front of the flame and quickly pass it through the flame in an outward motion to create a smooth hook or an L-shaped capillary tip.
Under a stereo microscope, manually pick the well-formed neuro retinal cups from the individual EFPs using the smooth outer curvature zone of the capillary hook as a fine scoop. At days 25 and 30, add four milliliters of pre-warmed retinal differentiation medium in a low attachment 60 millimeter dish to culture and maintain retinal cups for generating 3D retinal organoids. This should be done prior to harvesting the neuro retinal cups.
Set a one millimeter micropipette to aspirate 100 microliters and use one milliliter micro tips with wide bore openings to aspirate and transfer the floating retinal cups into the fresh low adherent culture dishes prepared earlier. Maintain the retinal cups in RDM as non-adherent suspension cultures and incubate them at 37 degrees Celsius in a 5%carbon dioxide incubator. On days 30 and 45, following a partial feeding method, remove half the volume of the spent medium and replace it with an equal volume of fresh medium.
On day 45 and 60, culture the retinal organoids for a further two weeks in RDM containing 100 micromolar taurine to support better survival and lineage differentiation of neuro retinal progenitors and the development of mature retinal cell types. Retinal organoids are characterized at different stages of maturation for the expression of several retinal progenitor markers using RT-PCR and immunohistochemistry. RT-PCR results confirmed the induction and expression of neuro retinal markers in one-month-old retinal organoids and two-month-old retinal organoids.
The confocal images of immuno-labeled sections of immature retinal organoids using antibodies against the neuro retinal progenitor markers Chx10, PAX6, and Otx2, and mature retinal organoids using antibodies against the photoreceptor precursor markers recoverin and CRX are shown here. The marked outermost layer of the retinal organoids with differentiating photoreceptor cells are zoomed and shown in these images. The rudimentary inner segment-like extensions are marked by white arrows.
EFP clusters with the neuro retinal cup at the center and migrating RPE outgrowths show pigmentation along the leading edges. Well-differentiated pigmented epithelial outgrowths from multiple EFPs all around the neuro retinal island are shown here. Higher magnification of an EFP shows the migratory zone of RPE progenitors and ocular surface epithelium surrounding a neuro retinal cup.
Extended adherent cultures that developed monolayers of immature RPE cells containing both pigmented and non-pigmented cells are shown here. Monolayer cultures of fully mature and pigmented RPE cells show cobblestone morphology at day 60. RPE cells expressing PAX6, MITF, and RPE65 are shown in this figure.
These images represent EFPs with abnormal aggregates of retinal progenitors with distorted lamination and lack of striations. EFP with the miniature neuro retinal cup, but lacking the surrounding zone of RPE and ocular surface epithelium are shown in this figure. The representative image shows irregular neuro retinal aggregates formed in the suspension culture.
It is very critical to start the differentiation process using near confluent growing cultures of IPCs to achieve efficient induction of eye fields within three to four weeks. Retinal organoids and RPE cells generated from normal and patient-specific IPCs can be used as retinal disease models and for testing novel therapeutic drugs.
