Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

The overall goal of this procedure is to reproducibly derive high quality retinal pigment epithelium, or RPE from stem cells. This is accomplished by first growing inducible pluripotent stem cells to co fluency. In the second step, the media is changed to direct the cells into RPE fates.

Next, the RPE cells are isolated from the other differentiated cells in the dish, and then the cultures are expanded until sufficient numbers of cells are obtained. Ultimately, the dark pigmentation and cobblestone morphologies of the cultures can be used to confirm the RPE cells are reaching terminal differentiation. The main advantage of this technique over existing methods like spontaneous differentiation, is that adding defined factors accelerates differentiation, time and increases RPE yield.

So to date, thousands of rodents have been cured using RPE cells, derived from IPSA variety of retinal degenerative diseases. However, if which we efficiently and effectively translate this into treating humans in the clinics with these diseases, it's absolutely imperative that we develop well-established reproducible protocols, which will provide us with high quality, high numbers of RPE sales and IPS Visual presentation of this method is essential is only one person can work in the microscope under the hood at any given time, making the isolation step difficult to visualize. Mitchell Prince, a research technician from my laboratory, will now be demonstrating the protocol associated with digital culture aspects of the procedure To direct the differentiation of stem cell derived RPE culture, the stem cell line of interest on a layer of mouse embryonic feeder cells at two times 10 to the six cells per well of a six well plate, allow the colonies to reach cof fluency and then switch to daily feedings with differentiation.

Medium supplemented with nicotinamide. After three weeks, begin adding active in a or IDE one to the feedings to enhance RPE differentiation. Two weeks later, switch back to the differentiation medium and nicotinamide alone discontinuing the daily feedings.

Once the pH indicators in the medium stop turning yellow the day after feeding, then return the culture to the incubator until eyelets of the pigmented cells appear that are large enough to be cut and removed. To isolate the pigmented eyelets next coat, the wells of a 24 well plate with a xeno free matrix that mimics the natural cell environment. Next, fill as many wells of the 24 well plate with differentiation media as needed.

And in a cell culture hood, use a dissecting scope and a scalpel to chop up each pigmented eyelet at the bottom of the original plate in two to six pieces and grab the pigmented parts with sharp forceps and then transfer one to two pigmented pieces into each.24. Well incubate the plate at 37 degrees Celsius and 5%carbon dioxide for three to five days until the cells adhere. Then begin changing the media three times per week.

Expand the cells for three to four weeks in the differentiation media. Continuing to feed the cells three times per week to passage. The RPE add 200 microliters of prewarm non trypsin cell dissociation solution at 37 degrees Celsius to detach the cells.

After five to eight minutes, stop the reaction with fresh differentiation media and then resuspend the cultures into single cell suspensions with a 200 microliter pipette, pooling the colonies with few cells. Next, transfer the dissociated colonies into 15 milliliter tubes and spin down the cells. Resus suspending the pellets in three milliliters of differentiation Media each transfer the colonies into each well of a matrix coded six well plate containing three additional milliliters of differentiation media per well.

Then expand the cells for one to two weeks until the cells are approximately 90%confluent and detach the cells again in one milliliter of dissociation solution as just demonstrated. This time. Using a 1000 microliter pipette to resuspend the cells, transfer the single cell solution to a 50 milliliter conical tube.

And then after spinning down the cells again res, suspend the pellet. In 18 milliliters of differentiation, media finally repl the cells in a matrix coded culture container to obtain the maximum number of desired cells in the minimum number of passages for further downstream analysis, pigmented colonies begin to appear after five to seven weeks of inducible pluripotent stem cell line culture. These colonies can continue to grow for weeks as the cultures are maintained.

Once they reach a sufficient size, they can be manually excised. Careful excision to avoid contamination with non RPE cells greatly facilitates the generation of sufficiently pure RPE cultures. Once mastered, large numbers of RPE cells can be derived in eight to 12 weeks if the technique is performed properly.

While attempting this procedure, it is important to remember to be careful not to contaminate the cultures while making media and feeding cells. After watching this video, our hope is that many of you will be able to produce large numbers of high quality RPE cells from IPS. Hopefully, this will facilitate more rapid translation from the bench to the bedside, and offer cell-based therapies to patients suffering from currently untreatable retinal degenerative neovascular diseases.