This protocol introduced a defined feeder cell free method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells. Limbal epithelial stem cells are responsible for renewing the corneal epithelial in the healthy eye. Damage to these cells leads to a condition known as limbal stem cell deficiency, and to the loss of corneal clarity.
The cell culture methods shown here enable efficient production of limbal epithelial stem cells for future corneal cell replacement therapies. To begin, passage and maintain the feeder free human pluripotent stem cell culture as outlined in the text protocol. Make sure to use only high quality human pluripotent stem cells as starting material for differentiations.
When ready to induce embryoid body formation, warm all of the needed materials and reagents to room temperature in a laminar flow hood. Detach the feeder free human pluripotent stem cells to the suspension by adding 500 microliters of xeno free trypsin EDTA to each well. An incubating at 37 degrees Celsius, with 5%CO2.
After three minutes, remove the cells from the incubator and check the cell morphology to determine the optimal phase for trypsin removal. Carefully observe the cells to determine the optimal time to remove the xeno free tryspin EDTA, when the cells have rounded up, but have not detached completely. At the optimal time, remove the xeno free trypsin EDTA from the cells.
Add defined trypsin inhibitor and gently pipette to detach the cells. Collect the single cell suspension in a 50 milliliter centrifuge tube. Centrifuge at 300g for five minutes.
Then, remove the supernatant, and resuspend the cells in one milliliter of culture medium. After counting the cells, distribute two to three million cells in three milliliters of basal induction medium supplemented with five micromolar blebbistatin to each well of a low attachment six well plate. Incubate overnight at 37 degrees Celsius with 5%CO2.
The next day, check the quality of the embryoid bodies under a microscope. Remove the medium, and replace it with three milliliters of basal induction medium supplemented with 10 micromolar SB-505124 and 50 nanograms per milliliter of human basic fiberglass growth factor. Continue incubating at 37 degrees Celsius with 5%CO2.
The following two days, remove the medium, and replace it with three milliliters of basal induction medium, supplemented with 25 nanograms per milliliter of bone morphogenetic protein four. Then, continue incubation using the previous conditions. On day four, prepare a mixture of 0.5 micrograms per square centimeter of Laminin-521, and five micrograms per square centimeter of human placental collagen type four, diluted in DPBS containing calcium two and magnesium two ions.
Using this mixture, coat the 100 millimeter tissue culture dishes for adherent differentiation in total coating volume of five milliliters per dish. Seal the plates, and store them at four degrees Celsius overnight. On day five, warm all of the needed materials and reagents to room temperature in a laminar flow hood.
After this, remove the coating solution and add 10 milliliters of pre-warmed differentiation medium to each dish. Using a pipette, transfer the embryoid bodies from a single plate well across two to three tissue culture dishes. Then, gently shake each culture dish to evenly distribute the embryoid bodies.
Maintain the cells in adherent culture at 37 degrees Celsius with 5%CO2 for the next two and a half to three weeks, making sure to replace the medium with 10 milliliters of fresh differentiation medium three times per week. Use a phase contrast microscope to regularly check the cells for the emergence of correct epithelial morphology. To begin, pre-warm all of the needed materials and reagents to room temperature in a laminar flow hood, except for the cryopreservation medium, which should be pre-chilled.
Next, detach the human pluripotent stem cell derived limbal epithelial stem cells to single cell suspension by adding xeno free trypsin EDTA and incubating at 37 degrees Celsius with 5%CO2. After five minutes, remove the cells from the incubator and check the cell morphology to determine the optimal phase for trypsin removal. Carefully observe the cells to determine the optimal time to remove the xeno free trypsin EDTA, when the cells have rounded up, but have not detached completely.
At the optimal time, remove the xeno free trypsin EDTA from the cells, and detach the cells as described earlier. After counting the cells, centrifuge them at 300g for five minutes. Aspirate the medium, and resuspend the cells in pre-chilled xeno free cryopreservation medium.
Using a pipette, transfer the single cell suspension into cryo-tubes so that each cryo-tube contains between 500, 000 to one million cells in one milliliter of cryopreservation medium. Place the tubes in a freezing container. Within five minutes, transfer them to a freezer at negative 80 degrees Celsius for overnight storage.
The next day, transfer the tubes to liquid nitrogen for long term storage. Before thawing the cells, coat all needed dishes and plate wells with a mixture of five micrograms per square centimeter human placental collagen type four, and 0.5 micrograms per square centimeter of LM-521, and pre-warm all of the needed materials and reagents to room temperature in the laminar flow hood. Next, remove the coating solution from the dishes and plate wells and add the appropriate volume of pre-warmed differentiation medium.
Add pre-warmed differentiation medium to a 15 milliliter conical tube. Thaw the cells quickly to room temperature. Once thawed, immediately transfer the cell suspension to the conical centrifuge tube.
Centrifuge at 300g for five minutes. Aspirate the medium, and resuspend the cell pellet in differentiation medium to remove any cryopreservation medium. Plate the cells onto precoated dishes in differentiation medium at a density of 40, 000 to 50, 000 cells per square centimeter.
Maintain the cells at 37 degrees Celsius at 5%CO2, replacing the differentiation medium three times a week. On Laminin-521, the undifferentiated high quality feeder free human pluripotent stem cells first form distinct colonies with sharp edges, which merge to homogenous monolayers upon confluence. Culturing in basal induction medium, supplemented with five micromolar blebbistatin for 24 hours typically produces a suspension of tight, regular embryoid bodies of various sizes, while the embryoid body morphology should not change dramatically during the surface ectodermal induction in suspension, colonial outgrowth is seen to appear soon after the embryoid bodies are plated onto the collagen type four and Laminin-521 combination matrix in differentiation medium.
Within 21 to 25 days of differentiation, the cells form confluent homogenous layers, with polygonal morphology, that is typical to epithelial cells. The cells may be then cryo stored for later use, as viability and morphology are well preserved after thawing. At day 24 of differentiation, the vast majority of the cells expressed paired box protein, PAX6, the key regulator of eye development, as well as p63 alpha, the widely recognized LESC marker.
Delta Np63 is coexpressed in most of the p63 alpha positive cells, confirming the most cornea specific delta Np63 alpha positive cell phenotype. Other markers are expressed in part, while others are undetectable at this point, indicating differentiation has progressed toward the unipotent limbal epithelial progenitors, but the terminal differentiation into mature corneal epithelial cells has not yet occurred. On average, each undifferentiated feeder free human pluripotent stem cell generates 0.7 cells by day 25.
At least 65%delta Np63 alpha positive cell population can be expected by day 24. Cryopreservation further purifies the cell population. Overall, the methods presented here are relatively simple, but there are a few critical points to success.
High quality of the starting material is essential, as well as gentle, fluent cell culture techniques in general. This protocol provides robust means to produce limbal epithelial stem cells for clinical applications, as well as for various research purposes. Moreover, the method can be easily modified for differentiation of retinal pigment epithelial cells.
