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.

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			<td height="21" style="height:21px;width:235px;">mTeSR1 media + 5X supplement</td>
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			<td></td>
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			<td height="18" style="height:18px;width:235px;">Non essential amino acids</td>
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			<td>SH3008803HI</td>
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			<td height="21" style="height:21px;">Triiodo-l-thyronine sodium salt</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">T6397</td>
			<td></td>
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			<td height="21" style="height:21px;width:235px;">Sodium hydroxide</td>
			<td style="width:156px;">Sigma</td>
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			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">195406</td>
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			<td height="21" style="height:21px;width:235px;">Phosphate buffered saline</td>
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			<td height="21" style="height:21px;width:235px;">Trypsin</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td style="width:119px;">25200-072</td>
			<td></td>
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			<td height="21" style="height:21px;width:235px;">Miltenyi Biotec washing buffer</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-092-987</td>
			<td></td>
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			<td height="21" style="height:21px;width:235px;">Miltenyi Biotec rinsing buffer</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-092-222</td>
			<td></td>
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			<td height="21" style="height:21px;width:235px;">Anti-TRA-1-60 microbead kit</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-095-816</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Miltenyi Biotec cell sorter column</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-021-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">RNeasy micro kit</td>
			<td style="width:156px;">Qiagen</td>
			<td style="width:119px;">74004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">QIA shredder</td>
			<td style="width:156px;">Qiagen</td>
			<td style="width:119px;">79654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">RNA 6000 Pico LabChip kit</td>
			<td style="width:156px;">Agilent</td>
			<td style="width:119px;">G2938-90046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Human miRNA microarray v16</td>
			<td style="width:156px;">Agilent</td>
			<td style="width:119px;">G4471A</td>
			<td></td>
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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...

Watch this Scientific Journal Video about MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium at JoVE.com

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

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, ...

microrna-expression-profiles-human-ips-cells-retinal-pigment

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11.3K Views.  JBSA Fort Sam Houston. 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. 

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

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

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.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Quantifying Retinal Pigment Epithelial Phagocytosis and Lipofuscin Accumulation

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

Author Spotlight: Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures  

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging ...