Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

The overall goal of this procedure is to characterize the influence of embryo body size on RPE differentiation from IPS cells for directing embryo body differentiation towards the RPE lineage. This is accomplished by first using the microwell technique to generate homogeneous embryo bodies of defined sizes. In the second step directed differentiation of the IPS cells is initiated towards the retinal lineage, and then the I-P-S-R-P-E cells are identified and isolated.

In the final step, the I-P-S-R-P-E cells are enriched and expanded. Ultimately, the I-P-S-R-P-E cells can be analyzed for their expression of I-P-S-R-P-E specific markers. The main advantage of this technique over existing methods like spontaneous differentiation, is that our technique results in higher of RP.This matter can help to optimize the directed differentiation of IPS cells towards the RP lineage.

The implications of this technique extend toward the therapy of retinal degenerative diseases such as macular degeneration, retinitis figment, or stargardt's disease. Since the replacement of diseased RPE with healthy RPE derived from IPS cells may be the best option for preventing loss of vision, generally Individual new to this method will struggle because stem cells are very delicate and subtle changes in their handle can affect the differentiation process. Visual demonstration of this method is critical as the embryo body harvesting step from the microwave place is difficult.

Prior to seeding warm the feeder free stem cell culture medium to 37 degrees Celsius, and then quickly warm the IMR 90 dash one IPS cells in a 37 degree Celsius water bath. When the cells are thawed, add five milliliters of warm feeder free stem cell culture, medium to the cells, dropwise and gently mix. Next, spin down the cells and then carefully resuspend the cell clumps in two milliliters of feeder free stem cell culture.

Medium seed the clumps in individual wells of a matrix coated plate, and then place the cells in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide and 95%humidity and daily medium changes. Five to seven days later, observe the undifferentiated colonies with a dense center that are ready for passage under a light microscope. When the cells are ready, add one milligram per milliliter of 37 degrees Celsius warm dis space to each well and incubate the plate at 37 degrees Celsius for seven minutes.

Then aspirate the DYS space and gently rinse the cell colonies with two milliliters of prewarm DM MF 12. Two times. After rinsing, add two milliliters of feeder free stem cell culture medium to each well, and use a five milliliter pipette to gently scrape the colonies, releasing the cell clumps.

Then transfer the detached clumps into a 15 milliliter conical tube and add sufficient feeder free stem cell culture medium to seed the next passage of cells to generate the embryo bodies. First, rinse the IPS cells with two milliliters of DMMF 12 and then add 750 microliters of Accutane to each well of the six well plate for five to 10 minutes. In a cell culture incubator, when the cells have begun to detach, use a pipette to gently dissociate the cells into a single cell suspension and then transfer them into a 50 milliliter conical tube.

Rinse the plate with five milliliters of DM MF 12, pooling the wash with the cells in the conical tube. Then pass the cell suspension through a 40 micrometer cell strainer to remove any residual cell clumps. Now spin down the cells to remove the Accutane and resus.

Suspend the pellet in embryo body formation medium to a 0.5 to one times 10 to the seventh cells per milliliter concentration. After determining the number of viable cells by trian blue exclusion, add the appropriate number of cells to each well of a micro well plate to generate the desired embryo. Body sizes.

Gently pipette the cultures to facilitate an even distribution of the cells, and then adjust the embryo body formation medium supplemented with rock inhibitor to a final volume of 2.0 milliliters. After more gentle pipetting, spin down the micro well cell cultures and incubate the plate in a cell culture incubator for 24 hours. The next day pipette medium up and down into each micro well with a one milliliter micro pipetter to harvest most of the embryo bodies and then pass the embryo body suspension through an inverted 40 micrometer cell strainer to remove any single cells.

Next, rinse the micro well plate five times with one milliliter of DMMF 12 to remove the rest of the embryo bodies passing the washes through the strainer. Then turn the cell strainer upside down in a Petri dish and filter the collected embryo bodies with more embryo body formation medium. After counting the collected cells incubate the embryo bodies on protein matrix coated six well plates at less than 1000 embryo bodies per well in embryo body formation medium, supplemented with 10 micromolar rock inhibitor in a cell culture incubator for 24 hours.

Finally, replace the embryo body formation medium with differentiation medium to initiate the differentiation. Collecting samples at day 6, 17, 29 and 60 to conduct the appropriate downstream analysis. After 12 weeks of culture, 200 cell embryo bodies develop astrocyte and fibroblast morphology with no visible pigmentation, whereas the larger embryo bodies develop a monolayer of classical RPE morphology and pigmentation as demonstrated in these images immunochemistry to detect the RPE markers, MITF and ZO one reveals the co-expression of these proteins derived from 500 cell and 3000 cell embryo bodies.

In this graph, the neuroectodermal marker PAC six was measured in differently sized embryo bodies at different time points by flow cytometry. For example, in the 3000 cell embryo bodies, approximately 50%of the analyzed cells were positive for PAC six on day six of culture. Additionally, fax analysis of the RPE marker MITF on embryo bodies of various sizes revealed that 20%of the cells expressed MITF by day 60 of differentiation.

Cultured RPE are also characterized by their ability to lose their pigment and polygonal morphology and to obtain a fibroblast phenotype upon packaging. For example, here, newly passage cells that have lost their pigment and have gained a fibroblast morphology are shown. These cells then proliferate, regaining their classical polygonal morphology upon confluence, and within a few weeks, regain their pigmentation.

While attempting this procedure, it's important to remember to properly identify and isolate the IPS RP colonies After its development. This technique paved the way for researchers in the field of stem cell biology to explore the directed differentiation towards many adult cell types by mimicking normal embryological development. Don't forget that working in the tissue, virtual labs can be extremely hazardous, and that repercussions, such as wearing leg coats, gloves, and glasses should always be taken while doing these procedures.

After watching this video, you should have a good understanding of how to generate homogeneous embryo bodies of the desire size to direct the deforestation towards I-P-S-R-P-E and to isolate and enrich RPE cells.