The overall goal of this procedure is to compare micro RNA expression in induced pluripotent stem cells. Retinal pigmented epithelial cells derived from induced pluripotent stem cells and fetal retinal pigmented epithelial cells. This is accomplished by first culturing and expanding IPS cells and fetal RPE.
The second step is to differentiate the IPS cells into RPE. Next, the RNA is extracted from the IPS cells, the I-P-S-R-P-E cells and the fetal RPE cells. The final step is to perform micro RNA microarray analysis on the RNA samples and analyze the data.
Ultimately, pathway and network analysis is used to identify differentially expressed microRNAs that are involved in multiple cellular networks, including proliferation and cell cycle progression. This method can help answer key questions in the vision research field, such as the role of micro RNA in regulating differentiation into mature RPE cell cycle progression, proliferation and senescence. The implication of this technique extend to therapy used in stem cell derived retina pigment epithelium to replace disease or damage RPE Because the Optima passage number of the cell used of therapy can be determined.
Though this matter can provide insight into the factors that regulate differentiation of RRP from IPS. It can also be applied to other organ systems such as differentiation of other TC specific cell types from IPS. To begin this procedure plate the induced pluripotent stem cells at about 350 cell colonies per well onto a newly coated plate in M tester one media, and allow the IPSL colonies to expand for four to five days in culture.
Then every three days replace the M tesser one media with four milliliters of differentiation media and change one half of the media with fresh differentiation media. Allow the pigmented foci to grow large enough to be manually dissected out of the culture, which takes about 40 to 50 days. Then use a 200 microliter pipette tip to cut around and lift the pigmented colonies.
Collect and pool the dissected colonies in a 15 milliliter conical tube. Add five milliliters of DMMF 12 media to the pooled RPE cells and centrifuge at 300 GS per five minutes. Next, remove the media re suspend the cell pellet in five milliliters of media and repeat centrifugation.
Remove the media from the cell pellet. Then prepare a single cell solution by incubating the pooled I-P-S-R-P-E with 0.25%TRIPSIN EDTA for five to 10 minutes, A 37 degrees Celsius until cell clumps are dissociated into single cells. Rinse the I-P-S-R-P-E cells with I-P-S-R-P-E media and then plate the collected cells on a fresh matrigel coated plate in four milliliters of I-P-S-R-P-E media culture, the cells for three weeks prior to passage and change the media with fresh I-P-S-R-P-E media every three days for the experiment.
See the I-P-S-R-P-E cells at 100, 000 cells per square centimeter and then collect the cells 17 days after seeding for collection of undifferentiated IPS cells. Culture, the cells until undifferentiated colonies are observed to be dense at the center. When this is observed, rinse the colonies in D-M-E-M-F 12 media, then dissociate the IPS cells into single cells using Accutane.
Collect the cells in a 15 milliliter conical tube and centrifuge at 300 Gs for five minutes. Aspirate the supernatant and resuspend the cell pellet in two milliliters of D-M-E-M-F 12 media to collect fetal RPE first rinse fetal RPE cell cultures with D-M-E-M-F 12. Then dissociate the cell cultures into single cells using 0.25%TRIPSIN EDTA for five to 10 minutes, A 37 degrees Celsius.
Collect the fetal RPE cells in a 15 milliliter conical tube and centrifuge at 300 GS for five minutes. Then aspirate the supernatant and resuspend the cell pellet. In two milliliters of D-M-E-M-F 12 media.
Collect the I-P-S-R-P-E cells on day 17. After passage three and passage five, rinse the I-P-S-R-P-E cells twice in PBS and prepare a single cell suspension by incubation with one milliliter of 0.25%trypsin for five to 10 minutes at 37 degrees Celsius. Then neutralize the trypsin with two milliliters of ice cold I-P-S-R-P-E media and collect the cells in a 15 milliliter conical tube.
Centrifuge the tube for five minutes at 300 Gs and then aspirate the snat. Re suspend the cell pellet in three milliliters of staining buffer from the microbead kit centrifuge, the I-P-S-R-P-E cell suspension in the staining buffer at 300 GS for five minutes. Then aspirate the supernatant and resus.
Suspend the cell pellet to a concentration of two times 10 to the six cells per 100 microliters of the staining buffer from the microbead kit. Add 10 microliters of anti TRA one 60 PE antibody per two times 10 to the six cells. Mix well and then incubate at four degrees Celsius for 10 minutes.
Wash the cells in one milliliter of staining buffer per two times 10 of the six cells. Then centrifuge the cell suspension for 10 minutes at 300 cheese. Next, aspirate the and resuspend the cell pellet in 80 microliters of staining buffer per two times 10 to the six cells.
Then add 20 microliters of anti PE labeled microbeads per two times 10 to the six cells. Mix well and incubate for 15 minutes at four degrees Celsius. Add one milliliter of staining buffer per two times 10 to the six cells centrifuge for 10 minutes at 300 Gs and aspirate the supernatant.
Then re suspend the cell pellet in 500 microliters of staining buffer and sort for TRA one 60 negative cells in a magnetic cell sorter as described in the accompanying text protocol ly the cells by running each sample through a commercially available micro centrifuge homogenizer mini spin column designed for nucleic acid mini preps. Then extract the total RNA from the IPS cells, I-P-S-R-P-E and fetal RPE with a commercially available RNA extraction kit. Designed to preserve small RNA molecules.
Use a NanoDrop spectrophotometer to determine the total RNA concentrations may make an Excel file with a list of the microRNAs that were identified to have at least twofold different expression levels between the sample groups. Then log into the online RNA pathways analysis program and upload the Excel file. Select flexible format for file format and select Agilent from the dropdown list for identifier.
Type under the raw data heading. Assign ID to the probe ID column and assign observation one and log ratio to the log ratio column. Select ignore for all other columns.
Select the parameters for analysis as follows, confidence experimentally observed only include direct and indirect relationships and include endogenous chemicals. Select the ingenuity knowledge base as the reference set. Then choose the database of reference molecules.
That includes data from all species, all cell lines and tissues and all mutations. After selecting all the parameters, run the network analysis by selecting networks. Then select overlapping networks and then click on a network of interest to see the results.
IPS cells that were differentiated into I-P-S-R-P-E cells exhibited the classic RPE phenotype of hexagonal pigmented cell morphology, which is similar to the fetal RPE cells comparison of the micro RNA expression profile of undifferentiated IPS cells to I-P-S-R-P-E cells revealed an upregulation in 47 microRNAs and 36 were downregulated, whereas the fetal RPE cells showed 61 microRNAs to be upregulated as compared to the I-P-S-R-P-E cells and 49 downregulated pathway analysis with ingenuity IPA software indicated the target genes of the differentially expressed microRNAs were involved in cellular processes including cellular development, growth and cell cycle. This analysis also indicated microRNAs expressed in ocular tissues in vivo were enriched in I-P-S-R-P-E and fetal RPE as compared to IPS cells. While attempting this procedure, it's important to remember to prevent contamination and degradation of the RNA samples following this procedure.
Other methods such as R-T-P-C-R can be performed in order to confirm the level of micro RNA expression in each cell type After its deployment. This technique paved the way for researchers in the field of vision research to explore the role of micro RNA in differentiation and cell cycle progression in rrp. After watching this video, you should have a good understanding of how to derive RPE from IPS and compare the microRNA expression profile of I-P-S-R-P-E to IPS cells and fetal RPE.
