Name: MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium (Video) | JoVE Journal
Uploaded: 2014-06-24T13:00:00.000+00:00
Duration: 10 min 19 s
Description: Learn microRNA profiling in human iPS cells and retinal pigment epithelium using microarray analysis. This method reveals regulatory networks in RPE development for regenerative medicine applications.

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5 minutes

The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
This research was performed while the authors Whitney A. Greene, Alberto Mu&#241;iz, and Ramesh R. Kaini held a National Research Council Postdoctoral Research Associateship at the USAISR.
Microarray assays were performed by the Greehey Children's Cancer Research Institute Microarray Core Facility and Bioinformatics Department at UT Health Science Center San Antonio.
This work was supported by U.S. Army Clinical Rehabilitative Medicine Research Program (CRMRP) and Military Operational Medicine Research Program (MOMRP).

1. Preparation of Culture Reagents and Culture Plates
<ol>
	<li>mTeSR1 media: Prepare mTeSR1 media according to the manufacturer&#8217;s directions. Thaw 100 ml of mTeSR1 5x supplement overnight at 4 &#176;C. Add the 100 ml of mTeSR1 5x supplement to 400 ml of mTeSR1 basal media and mix well. This media is stable at 4 &#176;C for up to 2 weeks and at -20 &#176;C for 6 months.</li>
	<li>Differentiation media: Prepare differentiation media in DMEM/F12 to yield 0.1mM &#946;-mercaptoethanol, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 10% knockout serum, and 10 &#181;g/ml gentamicin.</li>
	<li>iPS-RPE media: Prepare iPS-RPE media in MEM to yield 1x N1 supplement, 0.1 mM nonessential amino acids, 250 &#181;g/ml taurine, 13 ng/ml triiodo-L-thyronine sodium salt, 20 ng/ml hydrocortisone, 5 &#181;g/ml gentamicin, and 15% FBS26.</li>
	<li>Triiodo-L-thyronine sodium salt stock solution. To prepare stock solution dissolve 1.0 mg of triiodo-L-thyronine sodium salt in 1 N sodium hydroxide by gently swirling. Next add 49.0 ml of base media to make 50.0 ml of 20 &#181;g/ml of triiodo-L-thyronine sodium salt. Prepare aliquots and freeze at -20 &#176;C until needed. Use appropriate volume to achieve desired concentration in final culture media.</li>
	<li>Hydrocortisone stock solution: To prepare a hydrocortisone stock solution, solubilize 1 mg of hydrocortisone in 1 ml of absolute ethanol by gentle agitation. Next add 19.0 ml of base media for 20 ml hydrocortisone stock solution of 50 &#181;g/ml. aliquot and freeze at -20 &#176;C until needed. Use appropriate volume to achieve the desired concentration in the final culture media.</li>
	<li>Fetal RPE growth medium. Prepare the growth media by mixing all reagents in the RTEGM kit in the basal media, according to the manufacturer directions except for the FBS.</li>
	<li>Fetal RPE plating media. Prepare plating media by transferring a small amount of growth media to a sterile container and add FBS to a final concentration of 2%.</li>
	<li>Dispase: Prepare working solution of dispase of 1 mg/ml in DMEM/F12.</li>
	<li>Matrigel coated plates: Prepare matrigel in DMEM/F12 to 0.08 mg/ml.</li>
	<li>Coat each well of a 6-well plate with 1.0 ml of the matrigel solution.</li>
	<li>Incubate the coated plates at room temperature for 1.0 hr.</li>
	<li>After the 1.0 hr incubation, aspirate the excess DMEM/F12.</li>
	<li>Add 0.5 ml of mTeSR1 media to avoid drying. The matrigel coated plates are now ready for use.</li>
</ol>
2. iPS Culture
<ol>
	<li>Prior to iPS cell seeding have all tubes, warm mTESR1 media to room temperature, and the matrigel coated plates ready.</li>
	<li>Quickly thaw the IMR90-4 iPS cells in a 37 &#176;C water bath. Then remove the cryovial from the water bath and sterilize using 70% ethanol.</li>
	<li>Transfer the cells to a 15 ml conical tube using a 2 ml pipette to minimize breaking of cell aggregates.</li>
	<li>Drop-wise add 5 ml of warm mTeSR1 media to the cells and gently mix. Centrifuge the cells at 300 x g for 5 min at room temperature and remove the supernatant.</li>
	<li>Carefully resuspend the cell aggregates in 2 ml of mTeSR1 media and seed the cells onto one well of a six well plate.</li>
	<li>Place the plate into a 37 &#176;C incubator and culture with 5% CO2, 95% humidity.</li>
	<li>Change the iPS cell culture media daily. Undifferentiated colonies will appear 5-7 days after seeding. Check for undifferentiated colonies that are ready for passage (colonies with a dense center).</li>
</ol>
3. Passaging of iPS Cells
<ol>
	<li>Prior to beginning to passage the iPS cells, have all tubes, dispase solution, DMEM/F12 and mTERS1 media warmed to room temperature, and the matrigel coated plates ready.</li>
	<li>Before passaging iPS cells, remove any areas of differentiation by scraping the area and aspirating the media. Differentiation should remain below 20% of the well for high quality cultures. Carefully rinse the iPS cell-containing well with 2 ml DMEM/F12 and add 1 ml of 1 mg/ml dispase to each well.</li>
	<li>Incubate at 37 &#176;C for 7 min. Aspirate the dispase and gently rinse the cell colonies with 2 ml of DMEM/F12 twice.</li>
	<li>After rinsing, add 2 ml of mTSER1 to each well.</li>
	<li>Using a 5 ml pipette gently scrape the colonies of the plate to form aggregates.</li>
	<li>Transfer the detached colonies to a 15 ml conical tube. Add sufficient mTeSR1 media to seed the next passage of cells.</li>
</ol>
4. Differentiation of iPS Cells to iPS-RPE
<ol>
	<li>Plate the iPS cells on to a newly coated plate in mTESR1 media.</li>
	<li>Allow iPS cell colonies to expand for 4-5 days in culture.</li>
	<li>Substitute mTeSR1 media with 4 ml differentiation media. Change one half of the media every 3&#160;days.</li>
	<li>Allow pigmented foci to grow large enough to be manually dissected out of the culture (40-50 days).</li>
	<li>Use a 200 &#181;l pipette tip to cut around and lift the pigmented colonies.</li>
	<li>Collect and pool the dissected colonies in a 15 ml conical tube.</li>
	<li>Then add 5 ml of DMEM/F12 to the pooled RPE cells and centrifuge at 300 x g for 5 min twice.</li>
	<li>Prepare a single cell solution by incubating the pooled iPS-RPE with 0.25% trypsin EDTA.</li>
	<li>Rinse the iPS-RPE cells with iPS-RPE media and plate the collected cells on a fresh matrigel-coated well in 4 ml of iPS-RPE media.</li>
	<li>Culture the cells for 3 weeks prior to passage. Change the media every 3&#160;days. For the experiment, plate iPS-RPE at 100,000 cells/cm2. Collect cells on Day 17. Count the day of seeding as Day 0.</li>
</ol>
5. Sample Collection
<ol>
	<li>iPS Cell Collection
	<ol>
		<li>Culture the iPS cells until undifferentiated colonies are observed to be dense at the center.</li>
		<li>Rinse the colonies in DMEM/F12.</li>
		<li>Dissociate into single cells using accutase. Collect the cells into a 15 ml conical tube and centrifuge at 300 x g for 5 min. Aspirate supernatant. Resuspend in 2 ml of DMEM/F12.</li>
	</ol>
	</li>
	<li>Fetal RPE Collection
	<ol>
		<li>Rinse Fetal RPE cell cultures with DMEM/F12 and dissociate into single cells using 0.25% trypsin EDTA.</li>
		<li>Collect the cells in a 15 ml conical tube. Centrifuge at 300 x g for 5 min. Aspirate supernatant. Resuspend in 2 ml of DMEM/F12.</li>
	</ol>
	</li>
	<li>iPS-RPE Cell Collection
	<ol>
		<li>When the iPS-RPE reach passage 3 and passage 5, collect the cells on Day 17 after passage. Rinse iPS-RPE twice in PBS and prepare a single cell suspension by incubation with 1.0 ml of 0.25% trypsin.</li>
		<li>Neutralize trypsin with 2 ml of ice cold iPS-RPE media.</li>
		<li>Collect the cells in a 15 ml conical tube and centrifuge at 300 x g for 5 min. Aspirate supernatant.</li>
		<li>Resuspend the cell suspension in 3 ml of staining buffer from microbead kit.</li>
	</ol>
	</li>
</ol>
6. Negative Selection of iPS-RPE to Remove Undifferentiated Cells
<ol>
	<li>Keep all reagents at 4 &#176;C until use, but do not work on ice. Centrifuge cell suspension from step 5.3.4 at 300 x g for 5 min. Aspirate supernatant.</li>
	<li>Resuspend cells in 100 &#956;l of staining buffer from microbead kit per 2 x 106 cells.</li>
	<li>Add 10 &#956;l of Anti-TRA-1-60-PE antibody per 2 x 106 cells.</li>
	<li>Mix well and incubate at 4 &#176;C for 10 min.</li>
	<li>Wash cells in 1-2 ml of buffer per 2 x 106 cells. Centrifuge for 10 min at 300 x g. Aspirate supernatant.</li>
	<li>Resuspend cells in 80 &#956;l of staining buffer per 2 x 106 cells. Add 20 &#956;l of anti-PE labeled microbeads (from kit) per 2 x 106 cells. Mix well. Incubate for 15 min at 4 &#176;C.</li>
	<li>Add 1-2 ml of staining buffer per 2 x 106 cells. Centrifuge for 10 min at 300 x g. Aspirate supernatant.</li>
	<li>Resuspend cells in 500 &#956;l of staining buffer.</li>
	<li>Magnetic cell separation:
	<ol>
		<li>Place column in magnetic cell sorter. Rinse column with 3 ml of staining buffer.</li>
		<li>Apply cell suspension from 6.8 onto column.</li>
		<li>Place a tube at the negative port to collect the TRA-1-60 negative cells.</li>
	</ol>
	</li>
</ol>
7. Total RNA Extraction
<ol>
	<li>Lyse the cells by running the samples through a commercially available microcentrifuge homogenizer mini spin column designed for nucleic acid minipreps.</li>
	<li>Extract total RNA from iPS cells, iPS-RPE, and fetal RPE with the commercially available RNA extraction kit designed to preserve small RNA molecules27.</li>
	<li>Determine total RNA concentrations using a Nanodrop spectrophotometer.</li>
</ol>
8. Bioanalyzer and Microarray Assay
<ol>
	<li>Check the RNA integrity with Bioanalyzer in conjunction with commercially available RNA quality kit.</li>
	<li>Incubate 100 ng of total RNA with calf intestinal phosphatase at 37 &#176;C for 30 min</li>
	<li>Denature the RNA using 100% DMSO at 100 &#176;C for 5 min.</li>
	<li>Label the samples with pCp-Cy3 using T4 ligase by incubation at 16 &#176;C for 1 hr.</li>
	<li>Hybridize on an 8_x_15K format human miRNA array.</li>
	<li>Wash the arrays according to manufacturer&#39;s instructions and scan at a resolution of 5 &#181;m using a scanner.</li>
	<li>Acquire data using microarray feature extraction software compatible with the miRNA microarray.</li>
	<li>Process the miRNA signals to determine the relative level of expression in each sample.</li>
	<li>Compare the expression levels of each miRNA between the sample groups. Select miRNAs with at least two-fold differential expression for further analysis.</li>
	<li>Using commercially available software for data analysis, follow the instructions to generate heat maps for visualization of differentially expressed miRNAs28.</li>
</ol>
9. Pathway Analysis
<ol>
	<li>Save data from the miRNAs selected in 8.9 in Excel format.</li>
	<li>Log into the online RNA pathway analysis program. Upload the Excel file. Select &#8220;Flexible Format&#8221; for file format and select &#8220;Agilent&#8221; from drop down list for identifier type.</li>
	<li>Under raw data heading, assign &#8220;ID&#8221; to probe ID column and assign &#8220;Observation 1&#8221; and &#8220;log ratio&#8221; to the log ration column. Select &#8220;ignore&#8221; for all other columns.</li>
	<li>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. Choose the database of reference molecules that includes data from all species, all cell lines and tissues, and all mutations.</li>
	<li>Run network and pathway analyses.</li>
</ol>

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The authors have nothing to disclose.

In conclusion, this report describes the methods used to culture iPS cells, iPS-RPE, and fetal RPE. RPE derived from iPS are morphologically and functionally similar to fetal RPE. The iPS-RPE also expresses characteristic RPE genes including RPE65, CRALBP, PEDF, and LRAT16. To further characterize these cells, RNA was extracted and used to perform microarray analysis to identify differentially expressed miRNA. Analysis of the signal intensities revealed that the greatest number of differentially expressed miRN...

Stem cells have the capacity to replicate without limit and the potential to differentiate into any somatic cell type. The development of techniques to reprogram somatic cells into pluripotent stem cells has elicited great excitement in the research community and among clinicians, as the advent of personalized tissue regeneration is on the horizon1. Induced-pluripotent stem (iPS) cells exhibit the same features of unlimited replicative potential and pluripotency as embryonic stem (ES) cells while circumventing the ethical dilemmas associated with ESCs. In addition, patient-derived stem cells will not stimulate an immune response, greatly increasing the probability of successful therapeutic applications2-3. Under specific culture conditions, iPS cells have been shown to differentiate into several different cell types in vitro, including cardiomyocytes, neurons, pancreatic beta cells, hepatocytes, and retinal pigmented epithelium (RPE)4-12.
The RPE is a specialized layer of pigmented epithelial cells located at the back of the retina that performs several functions that are essential for visual health and function such as absorption of stray light, phagocytosis of the photoreceptor outer segments, and processing of retinoids for the production of visual chromophore. Dysfunction of the RPE due to damage or disease profoundly affects photoreceptor health and visual function as evidenced by the blinding diseases that are the result of underlying RPE pathology such as age-related macular degeneration (AMD), Stargardt&#8217;s disease, and retinitis pigmentosa (RP)13. Truly effective treatments that can restore vision have not been achieved, and replacement of diseased RPE with healthy RPE may be the best option to prevent loss of vision14-15. RPE derived from iPS (iPS-RPE) is a possible source of cells to replace the damaged RPE. iPS-RPE expresses characteristic RPE proteins LRAT, CRALBP, PEDF, and RPE65; displays the classical highly pigmented hexagonal RPE morphology; and performs RPE functions such as phagocytosis, retinoid processing, and secretion of 11-cis retinal5,16. However, before iPS-RPE can be used therapeutically, the iPS-RPE must be thoroughly characterized. Understanding the factors that govern RPE differentiation is necessary to improve the yield and purity of the cells to be used for clinical applications.
Cell differentiation is the result of highly regulated gene expression. Epigenetic remodeling of the genome and coordination of transcription factors are required for cell fate decisions that occur during differentiation and development17. Regulation of message RNA (mRNA) translation by microRNA (miRNA) presents yet another level of regulation that affects cell fate1. MiRNAs are short, ~22 nt, lengths of nucleotides that either suppress translation by binding to the 3&#8217; UTR of mRNA or target the mRNA for degradation. MiRNAs have been detected in virtually all tissues and to date over 2,000 unique human microRNAs have been registered in the miRBase database. Since miRNAs require only partial complementarity to bind to the target mRNA, a single miRNA can potentially bind to tens or hundreds of targets, and vice versa; i.e. a single mRNA can be targeted by several different miRNAs. This promiscuous binding characteristic dramatically increases the level of complexity of regulation as well as the level of difficulty of determining the functions of individual miRNAs and the role each plays in cellular functions18-21. Nevertheless, studies have shown that miRNA refines gene expression during differentiation by affecting DNA methylation status17. In a study more specific to the RPE, miR-204/211 was shown to promote the epithelial phenotype of the RPE22. Another group analyzed the miRNA profile of RPE during differentiation from ES cells and revealed distinct sets of miRNA are expressed during the differentiation process23. In fact, miRNA profiles can unambiguously distinguish cell types, including ES cells, precursor cells, and terminally differentiated cells24,25. Over 250 miRNAs are expressed in the retina. Based upon these studies, we hypothesize that miRNAs play an important role during the differentiation of RPE from iPS.
The objective of this report is to describe the protocols for the differentiation of RPE from IMR90-4 iPS cells, collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types, iPS cells, iPS-RPE and fetal RPE. Total RNA was extracted from cultures of each cell type and hybridized to a miRNA microarray, containing probes specific for 1,205 human miRNAs and 144 viral miRNAs. The microarray results were compared to determine which miRNAs were differentially expressed among the different cell types. miRNAs with 2 fold or greater fold change in expression were selected for further analysis. A miRNA analysis software program was used to identify potential targets of the differentially expressed miRNAs and to generate cellular networks regulated by the selected miRNAs.

<table><tbody><tr><td>mTeSR1 media + 5X supplement</td><td>Stem Cell Technologies</td><td>5850</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>11330-032</td><td></td></tr><tr><td>2-&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M-7154</td><td></td></tr><tr><td>Non essential amino acids</td><td>Hyclone(Fisher)</td><td>SH30853.01</td><td></td></tr><tr><td>Knockout serum replacement</td><td>Life Technologies</td><td>10828-028</td><td></td></tr><tr><td>Gentamicin&amp;nbsp;</td><td>Life Technologies</td><td>15750-060</td><td></td></tr><tr><td>MEM media</td><td>Life Technologies</td><td>10370-021</td><td></td></tr><tr><td>N1 supplement</td><td>Sigma</td><td>N-6530-5ML</td><td></td></tr><tr><td>Taurine</td><td>Sigma</td><td>T-8691-25G</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma</td><td>H0888-1G</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Hyclone(Fisher)</td><td>SH3008803HI</td><td></td></tr><tr><td>Triiodo-l-thyronine sodium salt</td><td>Sigma</td><td>T6397</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>S5881</td><td></td></tr><tr><td>Fetal RPE media RTEGM kit</td><td>Life Technologies</td><td>195406</td><td></td></tr><tr><td>Dispase</td><td>Life Technologies</td><td>17105-041</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354277</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Hyclone(Fisher)</td><td>10010-023</td><td></td></tr><tr><td>Trypsin</td><td>Hyclone(Fisher)</td><td>25200-072</td><td></td></tr><tr><td>Miltenyi Biotec washing buffer</td><td>StemGent</td><td>130-092-987</td><td></td></tr><tr><td>Miltenyi Biotec rinsing buffer</td><td>StemGent</td><td>130-092-222</td><td></td></tr><tr><td>Anti-TRA-1-60 microbead kit</td><td>StemGent</td><td>130-095-816</td><td></td></tr><tr><td>Miltenyi Biotec cell sorter column</td><td>StemGent</td><td>130-021-101</td><td></td></tr><tr><td>RNeasy micro kit</td><td>Qiagen</td><td>74004</td><td></td></tr><tr><td>QIA shredder</td><td>Qiagen</td><td>79654</td><td></td></tr><tr><td>RNA 6000 Pico LabChip kit</td><td>Agilent</td><td>G2938-90046</td><td></td></tr><tr><td>Human miRNA microarray v16</td><td>Agilent</td><td>G4471A</td><td></td></tr></tbody></table>

microrna expression profiles of human ips cells, retinal pigment epithelium derived from ips, and fetal retinal pigment epithelium

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

iPS cells (Figure 1A) were grown in differentiation conditions to induce differentiation into iPS-RPE cells. The iPS-RPE exhibited classic RPE phenotype of hexagonal pigmented cell morphology (Figure 1B) similar to fetal RPE (Figure 1C).
To better understand the role that miRNA may play during the process of differentiation from iPS to iPS-RPE, microarray analysis of miRNA expression was conducted. Total RNA was collected from iPS cells, fetal...

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

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.

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Research

JoVE Journal

Biology

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue.   This work has been extended to an animal model of several eye diseases.

 Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

Single-cell Profiling of Developing and Mature Retinal Neurons

Stem cell-derived retinal pigment epithelium  (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The objective of this report is to describe the protocols to derive the retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells using different sizes of embryoid bodies.

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

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Learn microRNA profiling in human iPS cells and retinal pigment epithelium using microarray analysis. This method reveals regulatory networks in RPE development for regenerative medicine applications.

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium (Video) | JoVE Journal

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

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