Name: differentiation, maintenance, and analysis of human retinal pigment epithelium cells: a disease-in-a-dish model for best1 mutations
Uploaded: 2018-08-24T12:30:00.000+00:00
Duration: 6 min 39 s
Description: Watch this Scientific Journal Video about Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations at JoVE.com

This project was funded by NIH grants EY025290, GM127652, and University of Rochester start-up funding.

1. Differentiation of hPSC to RPE
<ol>
	<li>Maintain and passage hPSCs as previously described18. 
	NOTE: All cells (including hPSCs and hPSC-RPEs) are grown in 37 &#176;C at 5% CO2 throughout the duration of the growth and differentiation protocols.</li>
	<li>Prior to differentiation, split confluent hPSCs to pre-coated 6-well plates.
	<ol>
		<li>To coat plates, thaw basement membrane matrix on ice for about 1 h, suspend liquidized matrix in 4 &#176;C DMEM medium at a 1:50 dilution, add 800 &#181;L coating mixture to each well, and incubate for at least 1 h at 37 &#176;C.</li>
		<li>When coating is complete, aspirate the mixture and immediately add 1.5&#8211;2 mL medium into the wells.</li>
		<li>To lift hPSCs from the old culture plates, incubate cells with PBS plus 0.5 mM EDTA for 5 min at room temperature, aspirate the solution, and resuspend cells in small clumps with culture medium.</li>
		<li>Seed cells at ~20% confluency in the new plates. 
		NOTE: Store basement membrane matrix in 100 &#181;L aliquots at -20 &#176;C and use one aliquot to coat a 6-well plate. The time for coating can be extended up to 4 h at 37 &#176;C, or overnight at room temperature. It is important to suspend and seed hPSCs as small clumps of 3&#8211;5 cells rather than single cells.</li>
	</ol>
	</li>
	<li>When cells are grown to full confluency, replace the culture medium with differentiation medium (4 mL/well) (Table 1, without activin-A). Culture for 14 days, changing the medium (4 mL/well) 3x/week. 
	NOTE: The hPSC plate density doubles approximately every 24 h. Significant cell death is expected after the start of the differentiation procedure. Aggregates of dead cells and debris in the wells can be seen by the naked eye during medium changes.</li>
	<li>From day 15 to day 28, supplement differentiation medium with 100 ng/mL human activin-A (Table 1, with human activin-A). Change the medium (4 mL/well) 3x/week.</li>
	<li>Stop activin-A supplementation starting on day 29 (Table 1, without human activin-A). Continue cell culturing in differentiation medium for 8&#8211;10 weeks until pigmented hPSC-RPE clusters appear (Figure 1A-B). 
	NOTE: Clusters of pigmented cells can be seen as small dark dots on a cell culture plate by the naked eye (Figure 1A-B, top). Individual pigmented cells with the signature cobblestone shape can be seen under a 20X microscope (Figure 1A-B, bottom).</li>
</ol>
2. Isolation and Culture of Differentiated RPE Cells
<ol>
	<li>Remove the differentiation medium, wash once with PBS, add 1 mL of 0.05% trypsin plus 1 U/&#181;L of collagenase into each well, and incubate at 37 &#176;C for 20&#8211;30 min. Remove the trypsin/collagenase and gently add 2 mL of pre-warmed RPE medium into each well of the 6-well plate (Table 2). 
	NOTE: Use an inverted microscope to monitor the morphology of the hPSC-RPE cells, which are polygonal before trypsin/collagenase treatment, and become round after sufficient digestion.</li>
	<li>Use a Bunsen burner to pull glass Pasteur pipettes.
	<ol>
		<li>With both hands, hold a long (9 inch) Pasteur pipette at both ends so it is horizontal, and above the Bunsen burner about 2/3 down the length of the thin part of the pipette.</li>
		<li>Once the glass becomes soft from the flame, quickly pull the two ends apart, such that a micro scraper-like tip is formed at the new end of the pipette (Figure 2).</li>
		<li>Repeat several times to create multiple &#34;cell scrapers&#34;. Spray the pulled pipettes with 70% ethanol for sterilization and air-dry them in the cell culture hood.</li>
	</ol>
	</li>
	<li>Under a microscope, use a micro &#34;cell scraper&#34; to gently dissociate pigmented hPSC-RPE clusters from the plate.
	<ol>
		<li>Gently tap (do not scrape) directly on the pigmented cells, which will float up into the culture medium once they dissociate from the bottom of the well.</li>
		<li>Immediately use a 20 &#181;L micropipette to gently suction the dissociated hPSC-RPE cells and transfer them to a pre-coated 6-well plate with RPE medium24.</li>
		<li>Collect ~5&#160;x 103 cells (~10% confluency as observed under a 20X microscope) in each pre-coated well of a 12-well plate by repeating steps 2.3.1&#8211;2.3.2 multiple times, as needed. Bring the final volume of medium to 2&#160;mL. 
		NOTE: To prevent dissociated hPSC-RPE cells from floating away, avoid movement of the plates during the dissociation process such that the dissociated cells are floating in the medium but still remain locally concentrated.</li>
	</ol>
	</li>
	<li>Culture the newly isolated hPSC-RPE cells (P0) on 12-well plates in RPE medium for another 6-8 weeks to allow them to form a pigmented monolayer (Figure 1C).</li>
	<li>Change the medium 3x/week. When changing the medium, leave approximately 1/3 volume of the old medium in each well, and add 2/3 volume of fresh, pre-warmed RPE medium. At this stage, use 2 mL of RPE medium for each well in a 12-well plate (thus, exchange 1.3&#160;mL of medium each time). 
	NOTE: The monolayer of P0&#160;hPSC-RPE&#160;cells may form domes, suggesting that the cells are actively transporting fluids, and are anchored by tight junctions25. Therefore, dome formation is an indicator of mature RPE status.</li>
</ol>
3. RPE Fate Validation by Immunoblotting
<ol>
	<li>Make the hPSC-RPE cell lysate using mammalian protein extraction buffer supplemented with proteinase inhibitor cocktail. Incubate the cell lysate on ice for 30 min before centrifugation at 13,000 x g for 15 min at 4 &#176;C to discard undissolved cell debris. Measure the total protein concentration of lysate.</li>
	<li>Denature 40 &#181;g of total protein in Laemmli SDS sample buffer at 75 &#176;C for 10 min. Separate the protein samples on a 10% Tris-glycine SDS-PAGE gel at a constant voltage of 90 V for 15 min and then 150 V until the dye front reaches the bottom of the gel.</li>
	<li>Transfer the proteins onto a nitrocellulose membrane in pre-cooled Tris-glycine buffer supplemented with 10% methanol at 100 V for 1 h.</li>
	<li>Block the membrane in Tris buffered saline with 0.1% non-ionic detergent (TBST) and 5% non-fat dry milk for 1 h at room temperature.</li>
	<li>Incubate the membrane with primary antibodies diluted in the blocking buffer at 4 &#176;C overnight. Dilute the antibodies targeting RPE specific marker proteins as follows: RPE65, 1:1,000; CRALBP, 1:1,000; BEST1, 1:1,000. Dilute the antibody against &#946;-actin at 1:10,000 to detect &#946;-actin as a loading control.</li>
	<li>Wash the membrane with TBST 3 times, with 5 min for each wash.</li>
	<li>Incubate the membrane with fluorophore conjugated goat anti-mouse IgG secondary antibody diluted 1:10,000 in blocking buffer for 1 h at room temperature.</li>
	<li>Wash the membrane with TBST 4 times, with 10 min for each wash.</li>
	<li>Detect the proteins using an infrared imaging system (Figure 3).</li>
</ol>
4. Checking BEST1 Subcellular Localization by Immunostaining
<ol>
	<li>Wash hPSC-RPE cells with 2 mL of PBS once and fix in 2 mL of 4% paraformaldehyde for 45 min at room temperature.</li>
	<li>Wash with 2 mL of PBS twice, and incubate the cells in 2 mL of PBS with 0.1% non-ionic surfactant and 2% donkey serum for 45 min to block the non-specific binding sites.</li>
	<li>Incubate the cells with BEST1 antibody (1:200 dilution) for 2 h at room temperature.</li>
	<li>Wash the cells with 2 mL of PBS 3 times. Incubate with fluorophore conjugated secondary IgG (1:1,000) for 1 h at room temperature.</li>
	<li>Wash with 2 mL of PBS 3 times to remove unbound secondary antibody.</li>
	<li>Observe stained cells by confocal microscopy (Figure 4).</li>
</ol>
5. Recording Ca2+-dependent Cl- Current in hPSC-RPE by Whole-cell Patch Clamp
<ol>
	<li>24&#8211;72 h before the patch clamp, split a fully confluent well (on a 12-well plate) of mature P0 hPSC-RPE cells (cobblestone-shaped and pigmented). Aspirate the medium, wash once with PBS, and add 1 mL of 0.05% trypsin plus 1 U/&#181;L collagenase into the well. 
	NOTE: Pre-warm the trypsin/collagenase to 37 &#176;C right before use.</li>
	<li>Incubate at 37 &#176;C for 8 min. 
	NOTE: After this step, the hPSC-RPE cells are still adherent and connected.</li>
	<li>Use a 1 mL micropipette to gently wash the cells off the plate, and transfer cells in the digestion mixture to a 15 mL conical tube. Wash the well with 1 mL of pre-warmed trypsin/collagenase to collect the residual cells and combine them into the 15 mL conical tube. 
	NOTE: The hPSC-RPE cells are still in large clumps at this point. Do not try to break the cell clumps by vigorous pipetting. Some clumps may stick to the inner wall of the 1 mL tip.</li>
	<li>Incubate the conical tube in a 37 &#176;C dry bath for 8 min.</li>
	<li>Use a 1 mL micropipette to gently pipette up-and-down 10&#8211;15 times the digestion mix. 
	NOTE: Cell clumps become much smaller but still visible. Avoid vigorous pipetting.</li>
	<li>Repeat the previous two steps. 
	NOTE: Most of the cells should be in single-cell suspension after pipetting. There may still be some tiny cell clumps, which are acceptable. Optional: Incubate the conical tube at 37 &#176;C for an additional 5 min followed by gentle pipetting. The total time in the trypsin/collagenase at 37 &#176;C should not exceed 30 min (8 + 8 + 8 + 5 = 29).</li>
	<li>Add 5 mL of pre-warmed RPE medium to the 15 mL conical tube containing the digested cells. Spin at 200 x g for 5 min to collect the cells. Re-suspend and seed cells on pre-coated 35 mm dishes at 10&#8211;20% confluency (e.g., a 100% confluent well on a 12-well plate to five 35 mm dishes for 10% confluency). 
	NOTE: At all times, avoid vigorous pipetting, which may cause significant cell death as represented by apparent dark cell debris in the supernatant after centrifugation. Well-separated single-cell seeding is essential for patch clamp recording.</li>
	<li>Perform the whole-cell patch clamp as described previously18,26,27,28,29,30,31. Acquire current traces from a family of step potentials (-100 to +100 mV from a holding potential of 0 mV) (Figure 5). 
	NOTE: The recipes of internal and external solutions are described in Table 3 and Table 4, respectively.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rem,+a+member+of+the+RGK+GTPases,+inhibits+recombinant+CaV1.2+channels+using+multiple+mechanisms+that+require+distinct+conformations+of+the+GTPase.">Rem, a member of the RGK GTPases, inhibits recombinant CaV1.2 channels using multiple mechanisms that require distinct conformations of the GTPase.</a> J Physiol. 588 (Pt 10), 1665-1681 (2010).</li><li>Gong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+Human+Protein-Induced+Pluripotent+Stem+Cells+toward+a+Retinal+Pigment+Epithelial+Cell+Fate.">Differentiation of Human Protein-Induced Pluripotent Stem Cells toward a Retinal Pigment Epithelial Cell Fate.</a> PLoS One. 10 (11), e0143272 (2015).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+activates+bestrophin+ion+channels+through+direct+interaction.">ATP activates bestrophin ion channels through direct interaction.</a> Nat Commun. 9 (1), 3126 (2018).</li></ol>

The authors have nothing to disclose.

The most important procedure for the disease-in-a-dish approach is to differentiate hPSCs with a disease-causing mutation to the correct cell lineage, which is RPE for BEST1. Thus, after each differentiation experiment, the resulting hPSC-RPE cells should be carefully validated for their mature status by RPE-specific morphologies and protein markers16,17,18. To minimize clonal artifact, multiple hiPSC clones from the same patient or multiple hESC clones with the same mutation should be used whenever possible.
Both hiPSC and hESC lines can be differentiated to RPE with the same protocol. hiPSCs with or without a mutation in the BEST1 gene can be reprogrammed from the skin fibroblasts of BEST1 patients or healthy donors, respectively. hESCs bearing a mutation in BEST1 can be genetically engineered from the parental hESC line, which has the wild-type (WT) BEST1. The hiPSC-RPE and hESC-RPE routes have their own advantages and disadvantages. The former is more patient-specific and clinically relevant, particularly to personalized medicine. However, it is worth noting that there are several limitations for the hiPSC-RPE approach: 1) it is logistically difficult to access a full spectrum of BEST1 patient samples, as donations from patients cannot always be granted, and some mutations are very rare in the first place; 2) non-specific phenotypes may result from different genetic backgrounds and physical conditions of the donors; 3) the hiPSC to RPE differentiation efficiency varies for different donors, such that some cases can be technically challenging to obtain hiPSC-RPEs. On the other hand, the hESC-RPE route, although more artificial, offers a versatile platform to generate isogenic hESC-RPEs with desired mutations on demand for non-biased functional investigations.
Mature RPE cells are pigmented, polygonal, and connected by tight junctions in a monolayer. After splitting into a single cell population for patch clamp, the freshly reseeded hPSC-RPE cells will lose the polygonal shape, but still retain pigmentation for several days (Figure 5A). Patch clamp is performed 24&#8211;72 h after cell splitting, during which time the pigmentation can still be used as a visible marker to select cells with good RPE status. A gentle cell split resulting in a majority of well-separated single cells without significant cell death is key to the success of the patch clamp.
Several other differentiation protocols using different cell culture media and growth factors have been documented in the literature21,32. The time required for differentiation of hPSC to RPE is similar in all these protocols including ours, while it is hard to tell if there is any significant difference in the differentiation efficiency without a side-by-side comparison. It should be noted that in the current protocol, hPSC-RPE cells are grown in regular flat bottom plates but not on plates with membrane inserts, so the hPSC-RPE generated by this procedure may not best recapitulate the polarity of RPE cells in vivo. Therefore, the techniques described above are mostly suited in a research setting33, although the generated hPSC-RPE cells can also be cultured in Transwell plates to form monolayer RPE sheets for clinical purposes17.

It has been documented that at least five retinal degenerative diseases are caused by genetic mutations in the BEST1 gene1,2,3,4,5,6,7,8, with the number of reported mutations already over 200 and still increasing. These BEST1-associated diseases, also known as bestrophinopathies, cause progressive vision loss and even blindness, and there are currently no effective treatments. The protein product of BEST1, namely BESTROPHIN1 (BEST1), is a Ca2+-activated Cl- channel (CaCC) specifically expressed in the retinal pigment epithelium (RPE) of the eyes5,6,8,9. Importantly, a clinical phenotype of BEST1-associated diseases is the reduced visual response to light stimuli, called light peak (LP) measured in the electrooculogram10,11; LP is believed to be mediated by a CaCC in RPE12,13,14. In order to better comprehend the pathological mechanisms of BEST1 mutations and to work towards potential therapies, it is essential to study mutant BEST1 channels endogenously expressed in human RPE cells.
However, obtaining RPE cells directly from live patients is highly impractical. Although native RPE cells can be harvested from biopsies of human cadavers and fetuses, the difficult accessibility to these sources significantly limits scientific research. Therefore, it is critical to have alternative RPE sources other than human eyes. This call has been answered by recent advancements in stem cell techniques, as functional RPE cells can now be differentiated from human&#160;pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), the latter being generated by reprogramming primary skin fibroblasts from donors16,17,18. Importantly, the self-renewal and pluripotency of hPSCs ensure a reliable source to generate RPEs, while the patient-specificity of hiPSCs and genomic modification potential of hESCs (e.g., by CRISPR) offer a versatile disease-in-a-dish model for desired BEST1 mutations.
hPSC-RPE has several advantages over mice RPE models: 1) BEST1 knockout mice do not display any retinal abnormality19, raising the possibility of different genetic requirement of BEST1 in RPE between mice and humans; 2) only 3% of human RPE cells are binucleate, in contrast to 35% in mice20; 3) hiPSC-RPE potentiates autologous transplantation in clinical treatment of retinal disorders21. Nevertheless, animal models are still indispensable for studying RPE physiology and pathology in a live system, and the oncogenic potential of hiPSC cannot&#160;be overlooked.
The procedure here describes a useful and moderately simple hPSC to RPE differentiation protocol that can be used for research and clinical purposes. This protocol uses nicotinamide (vitamin B3) to augment differentiation of hPSCs to neural tissue, which is further induced to differentiate into RPE by treatment with activin-A. Nicotinamide treatment has been shown to increase the number of pigmented cells (a sign of differentiation into RPE), possibly by attenuating the apoptotic activity of differentiating cells22. The resulting hPSC-RPE cells display the same key markers, cobblestone morphology, and cellular functionality as native human RPE cells22. Thus, in a research setting, the resulting hPSC-RPE cells are suitable for downstream functional analyses including immunoblotting, immunostaining, and whole-cell patch clamp, for which detailed experimental procedures are also provided. Clinically, RPE cells derived from stem cells have shown great potential for transplantation treatment of macular degeneration in both animal studies and human trials23.

<table><tbody><tr><td>Knock-Out (KO) DMEM</td><td>ThermoFisher</td><td>10829018</td><td></td></tr><tr><td>KO serum replacement</td><td>ThermoFisher</td><td>10829028</td><td></td></tr><tr><td>Nonessential amino acids</td><td>ThermoFisher</td><td>11140050</td><td></td></tr><tr><td>Glutamine</td><td>ThermoFisher</td><td>35050061</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>ThermoFisher</td><td>10378016</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>N0636</td><td></td></tr><tr><td>Human activin-A</td><td>PeproTech</td><td>120-14</td><td></td></tr><tr><td>MEM (a modification)</td><td>Sigma-Aldrich</td><td>M4526</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>97068-085</td><td></td></tr><tr><td>N1 supplement</td><td>Sigma-Aldrich</td><td>N6530</td><td></td></tr><tr><td>Glutamine-penicillin-streptomycin</td><td>Sigma-Aldrich</td><td>G1146</td><td></td></tr><tr><td>Nonessential amino acids</td><td>Sigma-Aldrich</td><td>M7156</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T0625</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0386</td><td></td></tr><tr><td>Triiodo-thyronin</td><td>Sigma-Aldrich</td><td>T5516</td><td></td></tr><tr><td>mTeSR-1 medium</td><td>Stemcell Technologies</td><td>5850</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356230</td><td></td></tr><tr><td>Collagenase</td><td>Gibco</td><td>17104019</td><td></td></tr><tr><td>Trypsin</td><td>VWR</td><td>45000-664</td><td></td></tr><tr><td>M-PER mammalian protein extraction reagent</td><td>Pierce</td><td>78501</td><td></td></tr><tr><td>proteinase inhibitor cocktail</td><td>Sigma-Aldrich</td><td>4693159001</td><td></td></tr><tr><td>RPE65 antibody</td><td>Novus Biologicals</td><td>NB100-355</td><td></td></tr><tr><td>CRALBP antibody</td><td>Abcam</td><td>ab15051</td><td></td></tr><tr><td>BEST1 antibody</td><td>Novus Biologicals</td><td>NB300-164</td><td></td></tr><tr><td>Beta Actin antibody</td><td>ThermoFisher</td><td>MA5-15739</td><td></td></tr><tr><td>Alexa Fluor 488-conjugated donkey anti-mouse IgG</td><td>ThermoFisher</td><td>A-21202</td><td></td></tr><tr><td>Goat anti-mouse IgG</td><td>ThermoFisher</td><td>SA5-35521</td><td></td></tr><tr><td>Goat anti-Rabbit IgG</td><td>LI-COR Biosciences</td><td>926-68071</td><td></td></tr><tr><td>Hoechst 33342</td><td>ThermoFisher</td><td>62249</td><td></td></tr><tr><td>HEKA EPC10 patch clamp amplifier</td><td>Warner Instruments</td><td>895000</td><td></td></tr><tr><td>Patchmaster</td><td>Warner Instruments</td><td>895040</td><td></td></tr></tbody></table>

differentiation, maintenance, and analysis of human retinal pigment epithelium cells: a disease-in-a-dish model for best1 mutations

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms remain elusive mainly due to the lack of a good in vivo model for studying BEST1 and its mutations under physiological conditions. BEST1 encodes an ion channel, namely BESTROPHIN1 (BEST1), which functions in retinal pigment epithelium (RPE); however, the extremely limited accessibility to native human RPE cells represents a major challenge for scientific research. This protocol describes how to generate human RPEs bearing BEST1 disease-causing mutations by induced differentiation from human pluripotent stem cells (hPSCs). As hPSCs are self-renewable, this approach allows researchers to have a steady source of hPSC-RPEs for various experimental analyses, such as immunoblotting, immunofluorescence, and patch clamp, and thus provides a very powerful disease-in-a-dish model for BEST1-associated retinal conditions. Notably, this strategy can be applied to study RPE (patho)physiology and other genes of interest natively expressed in RPE.

The most technically challenging step is the manual isolation, which aims to achieve a high purity of a differentiated P0 hPSC-RPE population. After a successful isolation, &#62;90% cells in the P0 population will grow and mature to display signature RPE morphologies (Figure 1C). The existence of a minor portion of non-RPE or immature RPE cells in the P0 population is almost inevitable, but will not interfere with the downstream experiments as long as the number of pigmented and cobblestone-shaped hPSC-RPE cells is the overwhelming majority.
With the hPSC-RPE based disease-in-a-dish approach, each patient-specific BEST1 mutation can be comprehensively characterized in vivo for its protein expression (immunoblotting, Figure 3), membrane trafficking (immunostaining, Figure 4), and ion channel function (whole-cell patch clamp, Figure 5). These results will provide critical information for elucidating the pathological mechanisms of the channel mutations, and for developing personalized medicine.
As BEST1 is predominantly expressed in RPE cells, it can be used as a cellular marker for the validation of a mature RPE status. It should be noted that some BEST1 mutations might affect its protein expression, so other well-established RPE markers such as RPE65 and CRALBP still need to be evaluated (Figure 3).
Matured P0&#160;hPSC-RPE cells can be maintained for 2-3 months before splitting (to P1) for whole-cell patch clamp. P0 cells older than 3 months are not recommended for patch clamp but can still be used for immunoblotting and immunostaining experiments.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57791/57791fig1.jpg" /> 
Figure 1: Differentiated hPSC-RPE cells at different stages. Representative images of pigmented hPSC-RPE clusters in culture plates at first appearance (A), before isolation (B), and after growth to maturity post isolation (C). Eye-view and 20X phase contrast images are shown in the top and bottom panels, respectively. <a href="/files/ftp_upload/57791/57791fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57791/57791fig2.jpg" /> 
Figure 2: Representative image of the micro cell scraper pulled from a Pasteur pipette. <a href="/files/ftp_upload/57791/57791fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57791/57791fig3v2.jpg" /> 
Figure 3: Validation of the mature RPE status of differentiated hPSC-RPE cells by immunoblotting. Blots showing the expression of RPE-specific protein markers BEST1, RPE65, and CRALBP in WT hPSC and hPSC-RPE cells. <a href="/files/ftp_upload/57791/57791fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57791/57791fig4v3.jpg" /> 
Figure 4: Immunostaining of BEST1 in WT hPSC-RPEs. (A) Representative immunofluorescent images showing the membrane localization of WT BEST1 in cobblestone-shaped hPSC-RPE cells. (B) Immunostaining negative control omitting the BEST1 primary antibody.&#160;
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57791/57791fig5v3.jpg" /> 
Figure 5: Whole-cell patch clamp recordings of hPSC-RPEs. (A) A single hPSC-RPE cell for whole-cell patch clamp. (B) Representative current traces recorded from a BEST1 WT hPSC-RPE and a BEST1 mutated hPSC-RPE at peak Ca2+. The voltage protocol used to elicit currents is shown in the Insert. Scale bar = 1 nA, 150 ms. See Tables 3 and Table 4 for patch clamp preparation details. <a href="/files/ftp_upload/57791/57791fig5v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>Knock-Out (KO) DMEM</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>KO serum replacement</td>
			<td>15% (75 mL)</td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>1% (5 mL)</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>1% (5 mL)</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin&#160;</td>
			<td>1% (5 mL)</td>
		</tr>
		<tr>
			<td>Nicotinamide&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Human activin-A*</td>
			<td>100 ng/mL</td>
		</tr>
		<tr>
			<td colspan="2">*Human activin-A is supplemented during days 15&#8211;28 of the differentiation protocol.</td>
		</tr>
	</tbody>
</table>
Table 1: RPE Differentiation Medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>MEM (&#945; modification)</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>5% (25 mL)</td>
		</tr>
		<tr>
			<td>N1 supplement</td>
			<td>1% (5 mL)</td>
		</tr>
		<tr>
			<td>Glutamine-penicillin-streptomycin</td>
			<td>1% (5 mL)</td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>1% (5 mL)</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>125 mg</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>10 &#181;g</td>
		</tr>
		<tr>
			<td>Triiodo-thyronin</td>
			<td>0.0065 &#181;g</td>
		</tr>
	</tbody>
</table>
Table 2: RPE Culture Medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>130 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Magnesium ATP</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES (pH 7.4)</td>
			<td>10 mM&#160;</td>
		</tr>
		<tr>
			<td>CaCl2*</td>
			<td>Varies</td>
		</tr>
		<tr>
			<td>Glucose**</td>
			<td>~ 5 mM</td>
		</tr>
		<tr>
			<td colspan="2">*Add CaCl2 to obtain desired Ca2+ concentration</td>
		</tr>
		<tr>
			<td colspan="2">** Use glucose to adjust osmolarity to 290&#8211;295 mOsm/L</td>
		</tr>
	</tbody>
</table>
Table 3: Patch Clamp Internal Solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>140 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES (pH 7.4)</td>
			<td>10 mM&#160;</td>
		</tr>
		<tr>
			<td>Glucose*</td>
			<td>~ 5 mM</td>
		</tr>
		<tr>
			<td colspan="2">*Use glucose to adjust osmolarity to 300&#8211;305 mOsm/L.</td>
		</tr>
	</tbody>
</table>
Table 4: Patch Clamp External Solution.

Watch this Scientific Journal Video about Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations at JoVE.com

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

differentiation-maintenance-analysis-human-retinal-pigment-epithelium

Nanyang Technological University

Research

JoVE Journal

Genetics

6.7K Views.  University of Rochester, School of Medicine and Dentistry. Hier presenteren we een protocol om te onderscheiden van de retinale pigment epitheel (RPE) cellen van menselijke pluripotente stamcellen rekening houdend met patiënt afkomstige mutaties. De mutant cellijnen kunnen worden gebruikt voor functionele analyses, met inbegrip van immunoblotting, immunofluorescentie, en patch klem. Deze aanpak van ziekte-in-a-schotel omzeilt de moeilijkheidsgraad van het verkrijgen van inheemse menselijke RPE cellen.

Video: Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

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.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Developmental Biology

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

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.

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Neuroscience

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

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

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.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.

In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority ...

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

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

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.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

Bioengineering

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Medicine

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Biology

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 ...

Assessing Retinal Thickness and Ganglion Cell Layer Counts in Rodent Eyeballs

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

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