This work was supported by the Garland Initiative for Vision, the California Institute for Regenerative Medicine (CIRM; grants DR1-01444, CL1-00521, TB1-01177, FA1-00616 and TG2-01151), The Vermont Community Foundation, The Breaux Foundation, and the Foundation Fighting Blindness Wynn-Gund Translational Research Acceleration Program.

1. Preparation of Reagents for Day 0 to Day 14 of the Protocol
<ol>
	<li>Prepare the following medium components:
	<ol>
		<li>Make 100 mL of retinal differentiation medium (RDM) by adding 1 mL of 100x N2 supplement, 2 mL of 50x B27 supplement, and 1 mL of 100x non-essential amino acid (NEAA) to 96 mL of Dulbecco&#39;s modified essential medium/nutrient mixture F12 9 (DMEM/F12).</li>
		<li>Make 10 mL of 1 M nicotinamide (NIC) by dissolving 1.221 g of NIC in 8 mL of sterile water, vortexing, and bringing the volume to 10 mL with sterile water. Sterile filter the solution.</li>
	</ol>
	</li>
	<li>Prepare the following growth factors and small molecules:
	<ol>
		<li>Reconstitute recombinant mouse noggin, human dickkopf WNT signaling pathway inhibitor 1 (DKK-1), and IGF-1 to 100 &#181;g/mL each in 0.1% bovine serum albumin (BSA) in phosphate-buffered solution (PBS). Aliquot as needed and store at -20 &#176;C for up to 3 months.</li>
		<li>Reconstitute FGF-basic to 10 &#181;g/mL and recombinant human/mouse/rat Activin A to 100 &#181;g/mL each in 0.1% BSA in PBS. Aliquot as needed and store at -80 &#176;C for up to 1 year.</li>
		<li>Reconstitute SU 5402 (FGF receptor-specific tyrosine kinase inhibitor) and CHIR99021 (glycogen synthase kinase 3, GSK-3&#946;, inhibitor) to 10 mM each in dimethyl sulfoxide (DMSO). Aliquot and store at -20 &#176;C for up to 1 year or 6 months, respectively.</li>
	</ol>
	</li>
	<li>Obtain the following for day 0 and/or day 14: 1x ethylenediaminetetraacetic acid (EDTA) solution (0.2 g EDTA per 1 L of PBS), 1X PBS -/- (PBS without calcium or magnesium, pH 7.4), 1x trypsin-like dissociation enzyme (TDE), DPBS (Dulbecco&#39;s PBS), RPE supporting medium (RSM), and Y-27632 dihydrochloride (use at 10 &#181;M).</li>
</ol>
2. Day 0: Day of Pluripotent Stem Cell Passage for Differentiation
<ol>
	<li>Grow stem cell colonies in feeder-free, serum-free conditions to approximately 80% confluence before passaging. 
	NOTE: See discussion for details on optimizing this step.</li>
	<li>Coat a 12-well plate with extracellular matrix-based hydrogel (ECMH) as per manufacturer recommendations. Allow to set for 1 h at room temperature or overnight at 4 &#176;C.</li>
	<li>Aliquot the volume of RDM and PBS -/- needed for day 0 and warm in a water bath to 37 &#176;C before adding growth factors . Bring EDTA to room temperature.</li>
	<li>Add the growth factors necessary for day 0 to the warmed RDM with 10 mM NIC, 50 ng/mL noggin, 10 ng/mL DKK-1, and 10 ng/mL IGF-1. From the stocks described in step 1.2, add 100 &#181;L of NIC, 5 &#181;l of noggin, 1 &#181;L of DKK-1, and 1 &#181;L of IGF-1 to 10 mL of RDM.</li>
	<li>Pick to remove all differentiated colonies based on morphology from the stem cells that will be passaged for differentiation. Use a P10 pipet tip to manually remove the differentiated cells. 
	NOTE: Fibroblastic cells between colonies as well as the opaque cells within colonies indicate differentiated cells to be removed. See discussion for details about differentiated cells.</li>
	<li>Passage a single well of a 6-well plate into 4 wells of a 12-well plate (1:4). 
	NOTE: See discussion for details on passaging stem cells at this stage.
	<ol>
		<li>Aspirate the stem cell medium from the stem cells and wash the wells once with 2 mL of pre-warmed PBS -/-.</li>
		<li>Aspirate PBS -/- and rinse each well three times with 1 mL of EDTA per well of a 6-well plate.</li>
		<li>Gently tilt the plate and aspirate the EDTA. Do not agitate the plate in any way to avoid prematurely lifting the cells.</li>
		<li>After the third wash, add 1 mL of EDTA and incubate at room temperature in the hood for 3-5 min. Do not disturb the plate during this incubation.</li>
		<li>Aspirate the EDTA and add 1 mL of RDM per well that will be seeded with 0.5 mL of extra medium. For example, wash 1 well of a 6-well plate with 4.5 mL of RDM to plate on 4 wells of a 12-well plate.</li>
		<li>Use a cell scraper to gently detach the cells. Collect all cells in a conical tube and triturate the cells in RDM by pipetting up and down 5 times. Dissociate large clumps of cells, but do not triturate to single cell suspension. Distribute the cells evenly in the pipet. Complete this step quickly to prevent reattachment to the plate.</li>
		<li>Seed cells on the ECM-coated 12-well plates (1 mL of cell suspension per well).</li>
		<li>Tilt the plate back and forth to distribute the cells evenly throughout the wells. Gently place in a cell culture incubator at 37 &#176;C and 5% CO2 until the next medium change.</li>
		<li>Note the exact time. Change medium at the same time each day.</li>
	</ol>
	</li>
</ol>
3. Day 1 to 14: Addition of Growth Factors
<ol>
	<li>Day 1: Change the medium on all wells (1 mL per well) using RDM with the growth factor composition for day 0 (see step 2.4).</li>
	<li>Day 2: Change the medium using RDM (1 mL per well) with 10 mM NIC, 5 ng/mL FGF-basic, 10 ng/mL noggin, 10 ng/mL DKK-1, and 10 ng/mL IGF-1. From the stocks described in step 1.2, add 100 &#181;L of NIC, 5 &#181;L of FGF-basic, 1 &#181;L of noggin, 1 &#181;L of DKK1, and 1 &#181;L of IGF1 to 10 mL of RDM.</li>
	<li>Day 4: Change the medium using RDM (1 mL per well) with 100 ng/mL activin A, 10 ng/mL DKK-1, and 10 ng/mL IGF-1. From the stocks described in step 1.2, add 10 &#181;L of activin A, 1 &#181;L of DKK1, and 1 &#181;L of IGF-1 to 10 mL of RDM. 
	NOTE: Observe that cells are confluent at this stage.</li>
	<li>Day 6: Change the medium using RDM (1 mL per well) with 100 ng/mL activin A and 10 &#181;M SU 5402. From the stocks described in step 1.2, add 10 &#181;L of activin A and 10 &#181;L of SU 5402 to 10 mL of RDM.</li>
	<li>Days 8, 10, and 12: Change the medium using RDM (1 mL per well) with 100 ng/mL activin A, 10 &#181;M SU 5402, and 3 &#181;M CHIR99021.From the stocks described in step 1.2, add 10 &#181;L of activin A, 10 &#181;L of SU 5402, and 3 &#181;L of CHIR99021 to 10 mL of RDM.</li>
</ol>
4. Day of Enrichment to Passage 0 of RPE
<ol>
	<li>Coat a 6-well plate with growth factor reduced ECMH as per manufacturer recommendations. Allow to set for 1 h at room temperature or overnight at 4 &#176;C.</li>
	<li>Aliquot the volume of DPBS needed and 1 mL of RDM per well of enrichment and warm in a water bath to 37 &#176;C. Bring the TDE to room temperature and warm necessary volume of RSM, antimicrobial reagent, and Y-27632 to 37 &#176;C.</li>
	<li>Add antimicrobial reagent and Y-27632 obtaining 0.5x and 10 &#181;M compositions respectively to RSM. Use this medium for the first 4-7 days to improve attachment.</li>
	<li>Aspirate spent medium from all wells and add 1 mL per well of pre-warmed RDM (no growth factors required).</li>
	<li>Using a dissecting microscope, manually dissect and scrape away all non-RPE cells using a P10 pipet tip. 
	NOTE: See the representative results section for examples.</li>
	<li>After dissection, aspirate RDM and all cell debris. Wash twice with 1 mL of pre-warmed DPBS per well.</li>
	<li>Add 0.5 mL of TDE per well of a 12-well plate and incubate at 37 &#176;C for 5 min. Use a cell scraper to gently remove the cells from the plate. Use a P1000 pipet to gently triturate the cell/TDE suspension by pipetting up and down 3-4 times to create a uniform suspension.</li>
	<li>Dilute the cell/TDE suspension 1:10 in pre-warmed RSM, without Y-27632. Centrifuge cell suspension at 173 x g for 5 min at room temperature.</li>
	<li>Aspirate the medium from the cell pellet and resuspend the cells in RSM with 10 &#181;M Y-27632 (1 mL per enriched well).</li>
	<li>Strain the cells using a nylon mesh cell strainer with 40 &#181;m pores. Count the number of cells in a specified volume using a hemocytometer and calculate the concentration of cells in the strained solution.</li>
	<li>Seed cells on the growth factor reduced ECM-coated plates at 1 x 105 cells/cm2 in 4 mL of RSM with 10 &#181;M Y-27632.</li>
	<li>Replace the RSM with 10 &#181;M Y-27632 48 h after cell seeding and continue to replace media every 3-4 days (e.g. on Mondays and Thursdays). Do not replace the 10 &#181;M Y-27632 after 4-7 days.</li>
	<li>Allow the cells to mature for 28 to 35 days at 37 &#176;C and 5% CO2. Continue to replace the RSM every 3-4 days (e.g. on Mondays and Thursdays).</li>
</ol>
5. Maturation: Passage 1 and 2 of RPE
NOTE: volumes are indicated for 1 well of a 6-well plate or a T75 flask as indicated by parentheses.
<ol>
	<li>Between days 28 to 35 of passage 0, coat a 6-well plate (T75 flask) with ECMH as per manufacturer recommendations.</li>
	<li>Aliquot the volume of DPBS and RSM needed and warm in a water bath to 37 &#176;C. Bring TDE to room temperature.</li>
	<li>Aspirate spent medium from wells and wash each well twice with 2 mL (10 mL) of pre-warmed DPBS. 
	NOTE: Do not use 10 &#181;M Y-27632.</li>
	<li>Aspirate DPBS and add 1 mL (5 mL) of TDE. Place in incubator at 37 &#176;C and 5% CO2 for 5 min. After incubation, view cells on an inverted microscope to confirm the cells are contracting and detaching.</li>
	<li>Using an appropriately sized cell scraper, gently remove the cells from the bottom of the well or flask.</li>
	<li>Use a P1000 tip (10 mL serological pipet) to gently triturate the cell/TDE suspension up and down 3-4 times to create a uniform suspension.</li>
	<li>Dilute cell suspension 1:10 in RSM. Reserve 2 mL (5 mL) of RSM to rinse the well/flask and add to the diluted cell suspension. 
	NOTE: Do not allow enzyme exposure time to exceed 25 min.</li>
	<li>Centrifuge the cell suspension at 173 x g for 5 min at room temperature.</li>
	<li>Aspirate the medium from the cell pellet and resuspend the cells in 1 mL (5 mL) of RSM</li>
	<li>Strain the cells using a nylon mesh cell strainer with 40 &#181;m pores. Count the number of cells in a specified volume using a hemocytometer and calculate the concentration of cells in the strained solution.</li>
	<li>Seed cells on the ECMH-coated plates at 1 x 105 cells/cm2 in 4 mL (15 mL) of RSM.</li>
	<li>Allow the cells to mature for 30 days. Continue to change the RSM every 3-4 days.</li>
	<li>Repeat the above procedure (step 5.2-5.11) at day 30 to passage the cells from passage 1 to 2.</li>
</ol>
6. Creating an Intermediate Cell Bank: Cryopreservation of Passage 2 day 3-5 RPE
NOTE: Cryopreserve cells while they are subconfluent (~50%) and have not regained pigment.
<ol>
	<li>Based on the number of cells, calculate the volume of cryopreservation medium with 10% DMSO needed to resuspend the cells at a concentration of 3x106 cells/mL.</li>
	<li>Follow steps 5.2 to 5.8. Resuspend the cell pellet in the cryopreservation medium with 10% DMSO to 3 x 106 cells/mL and transfer 1 mL of the cell suspension to 1.2 mL cryogenic vials.</li>
	<li>Place cryogenic vials in a freezing container designed to cool at -1 &#176;C/min and place at -80 &#176;C overnight. Transfer to liquid nitrogen for long-term storage. 
	NOTE: These cells will be passage 3 upon thawing. Culture the cells for 30 more days before characterization. Seed passage 3 RPE at 1.5 x 105 per cm2 upon thawing.2,4,6,7</li>
</ol>

<ol>
	<li>Strauss, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+pigment+epithelium+in+visual+function.">The retinal pigment epithelium in visual function.</a> Physiol. Rev. 85 (3), 845-881 (2005).</li><li>Leach, L. L., Clegg, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+Review:+Making+Stem+Cells+Retinal:+Methods+for+Deriving+Retinal+Pigment+Epithelium+and+Implications+for+Patients+With+Ocular+Disease.">Concise Review: Making Stem Cells Retinal: Methods for Deriving Retinal Pigment Epithelium and Implications for Patients With Ocular Disease.</a> Stem Cells. 33 (8), 2363-2373 (2015).</li><li>Pennington, B. O., Clegg, D. 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Dr. Clegg is a co-founder of Regenerative Patch Technologies LLC.

This protocol describes how to produce retinal pigment epithelial cells from pluripotent stem cells. The method was optimized using both human embryonic and induced pluripotent stem cells from a feeder-free, serum-free culture method. Since the initial isolation of human embryonic stem cells in 1998 and the derivation of induced pluripotent stem cells (iPSC) in 2007, a multitude of stem cell culture methods have been developed14,15,16,17. These methods should be sufficient for producing stem cell colonies that are susceptible to this differentiation. There are no known limitations to the applicability of this method to properly derived and maintained pluripotent stem cells.
The most critical steps are the passaging of stem cells to day 0 of differentiation (step 2.5) and the potential need for manual dissection at day 14 of the process (step 4.5). When picking to remove differentiated cells from the stem cell colonies, refer to the images in Kent18. As indicated, the fibroblastic cells between colonies and the opaque cells within colonies indicate differentiated cells that need to be removed before beginning this protocol18. Only undifferentiated, tightly packed colonies with defined edges should be passaged for differentiation.
The number of stem cells seeded per well (step 2.6.7) is complicated by the fact that the stem cells cannot be triturated into a single cell suspension upon passage and cannot be accurately counted using a hemocytometer. The approximation of 80% confluent stem cells is indicated for passaging 1 well of a 6-well plate into 4 wells of a 12-well plate. Differences between stem cell lines, such as growth rate, can affect how quickly the immature RPE reach confluence between days 0 to 4. The stem cells will produce RPE regardless of precise confluence, but the cell yield will be negatively affected if the cells are too sparse at this stage. The immature RPE cells should be approximately 40-50% confluent on day 1 and nearly 100% confluent by day 4. If the cells are not producing a confluent monolayer by day 4 or 6, the protocol should be repeated at a higher seeding density at day 0. For example, if 1 well of a 6-well plate was passaged to 4 wells of a 12-well plate at day 0 and the immature RPE are not 100% confluent at day 4, reduce the seeding to a 1:3 or 1:2 passage on day 0 or allow the stem cells to become more confluent before passaging. It is critical to establish a consistent seeding density when comparing multiple cell lines.
The manual dissection step at day 14 is only necessary when non-RPE cells are present in culture (Figure 2C). Since the addition of CHIR99021 to the protocol, many pluripotent stem cell lines require little to no manual dissection. Some preparations have a higher incidence of neural patches and it is critical to remove those cells. If the RPE are not viable at passage 0 through passage 3, it is possible to repeat the differentiation protocol taking sufficient time to remove all non-RPE cells. This does not happen often, but it is mentioned here to note that the dissection step on day 14 can be optimized when needed.
There are a variety of RPE differentiation protocols that vary in cost as well as culture methods, efficiency, quantification, and functional assessment, the latter of which has been reviewed thoroughly2. We prefer the 14-day method detailed here because of its efficacy, adaptability, and applicability to a wide range of cell lines4,7,8. The cryopreservation step in this protocol also provides a major advantage in creating an intermediate cell bank for future use, avoiding lot-to-lot variability in experiments. Starting with only 4 wells of a 12-well plate, it is possible to expand into 6-well plates at passage 0 and T75 flasks at passage 1 and 2. At passage 2 day 3-5, when the cells are still subconfluent and have not regained pigment, it is possible to cryopreserve tens of millions of cells and then thaw the mature RPE, designated passage 3 day 30, to check RNA expression, protein expression, growth factor secretion, phagocytosis, etc. We have also established protocols to expand RPE for up to 13 passages 19.
Looking forward, this method will be useful for iPSC modeling of ocular disease and for generation of RPE for cellular therapy. With regards to iPSC disease modeling, this protocol is currently being used in the lab to produce RPE from CRISPR-corrected lines with non-corrected controls from the same patient. Furthermore, this protocol is adaptable to synthetic substrates and xeno-free conditions that are useful for adhering to the good manufacturing practices required for a cellular therapy.

The retinal pigment epithelium is a monolayer of pigmented cells that provides crucial support for photoreceptors. Retinal pigment epithelial cells (RPE) have numerous functions in vision, including light absorption, nutrient and ion transport, retinoid cycling, photoreceptor outer segment phagocytosis, and growth factor secretion1. There are a variety of retinal dystrophies that affect the function of RPE and result in a loss of vision, including age-related macular degeneration and retinitis pigmentosa. Generation of RPE from pluripotent stem cells may facilitate research to understand these eye diseases, and can provide an unlimited source of RPE for cell therapies2. In fact, multiple clinical trials are underway using RPE derived from pluripotent stem cells3.
This differentiation protocol was originally described by Buchholz4 and was based on the previously published method from Clegg5. The procedure mimics the normal in vivo developmental process to direct undifferentiated pluripotent stem cells towards an RPE fate via manipulation of the insulin growth factors (IGF), basic fibroblast growth factor (FGF-2; FGF-basic), transforming growth factor beta (TGF-&#946;), and WNT pathways4,5. The protocol was significantly improved by addition of a WNT pathway agonist late in the protocol, which yielded 97.77% &#177; 0.1% pre-melanosome protein (PMEL) positive cells, and has been adapted to xeno-free conditions6,7. The resulting RPE have been shown to express RPE markers at the transcript and protein levels, to secrete known RPE growth factors with appropriate polarity, and carry out phagocytosis of photoreceptor outer segments8. This protocol is more rapid and reliable than &#34;spontaneous&#34; protocols of differentiation that involve simple removal of basic fibroblast growth factor8. Furthermore, RNA sequencing data show that RPE obtained using this protocol are very similar to those obtained using the more common spontaneous approach8. The 14-day method generates RPE that fit the &#34;5 P&#39;s&#34; mentioned by Mazzoni9 (pigmented, polarized, phagocytic, post-mitotic, polygonal)9. While this procedure has proven to be reproducible in multiple labs, we wish to acknowledge several additional directed differentiation methods that have been published in recent years10,11,12,13.

<table><tbody><tr><td>SterilGARD III laminar flow biosafety cabinet</td><td>Baker</td><td>model: SG603A-HE, type: A2, class: 2</td><td>6&#039; Baker laminar flow biosafety cabinet</td></tr><tr><td>Dissection Hood</td><td>Labconco</td><td>Model 3970405</td><td>laminar flow bench top</td></tr><tr><td>dissecting microscope</td><td>Nikon</td><td>SMZ 1500</td><td>heated stage</td></tr><tr><td>air-jacketed CO2 incubator</td><td>Sanyo</td><td>MCO-17AIC</td><td>37 oC and 5% CO2</td></tr><tr><td>inverted phase contrast microscope</td><td>Olympus</td><td>IX53</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Media Components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>10565042</td><td></td></tr><tr><td>N2 Supplement</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>B27 Supplement</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>NEAA</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Growth Factors and Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>Recombinant mouse noggin</td><td>R&amp;amp;D systems</td><td>1967-NG-025</td><td></td></tr><tr><td>Recombinant human DKK-1</td><td>R&amp;amp;D systems</td><td>5439-DK-010</td><td></td></tr><tr><td>Recombinant IGF-1</td><td>R&amp;amp;D systems</td><td>291-G1-200</td><td></td></tr><tr><td>FGF-basic</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>Recombinant human/mouse/rat Activin A</td><td>Peprotech</td><td>120-14E</td><td></td></tr><tr><td>SU5402 FGF inhibitor</td><td>Santa Cruz Biotechnology</td><td>sc-204308</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Substrates&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Matrigel Basement Membrane Matrix, Phenol Red-Free, LDEV-Free</td><td>Corning</td><td>356237</td><td>extracellular matrix-based hydrogel (ECMH)</td></tr><tr><td>Matrigel hESC-Qualified Matrix, LDEV-Free</td><td>Corning</td><td>354277</td><td>growth factor reduced ECMH</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1X Versene (EDTA)</td><td>Gibco</td><td>15040066</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190250</td><td></td></tr><tr><td>1X PBS (no calcium, no magnesium)</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>TrypLE (trypsin-like dissociation enzyme, TDE)</td><td>Gibco</td><td>12563011</td><td></td></tr><tr><td>X-VIVO 10 (RPE supporting medium)</td><td>Lonza</td><td>BW04-743Q</td><td></td></tr><tr><td>Y-27632</td><td>Tocris</td><td>12-541-0 (1254)</td><td></td></tr><tr><td>CryoStor CS10</td><td>BioLife Solutions</td><td>210102</td><td>cryopreservation medium</td></tr><tr><td>1.2 mL Cryogenic Vial</td><td>Corning</td><td>430487</td><td></td></tr><tr><td>Mr. Frosty (freezing container)</td><td>Nalgene</td><td>5100-0001</td><td>freezing container</td></tr><tr><td>Normocin</td><td>Invivogen</td><td>ant-nr-2</td><td>antimicrobial reagent</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pipet-aid</td><td>Drummond</td><td>4-000-101</td><td></td></tr><tr><td>12-well culture plate</td><td>Corning</td><td>CLS3516</td><td>Used during differentiation.</td></tr><tr><td>T75 flask</td><td>Corning</td><td>430641</td><td>Used during RPE maturation.</td></tr><tr><td>6-well culture plate</td><td>Corning</td><td>CLS3513</td><td>Used during RPE maturation.</td></tr><tr><td>cell scraper</td><td>Corning</td><td>08-771-1A</td><td>Used during passages.</td></tr><tr><td>cell strainer</td><td>Falcon</td><td>352340</td><td>Used during passages before cell count.</td></tr></tbody></table>

rapid, directed differentiation of retinal pigment epithelial cells from human embryonic or induced pluripotent stem cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This method results in the production of a homogeneous, pigmented, and cuboidal monolayer of RPE. The timeline in Figure 1 corresponds to the images depicted in Figure 2. As shown in Figure 2A, the stem cell colonies are tightly packed with defined edges and no fibroblastic cells between colonies or opaque cells within colonies. Figure 2B provides a representation of immature RPE that are subconfluent. If the cells are already confluent at this stage, they cannot extend projections that are critical to the differentiation process. Cells that are severely subconfluent will not be able to establish a monolayer and form tight junctions, characteristic of epithelium. Details on how to optimize this confluence are outlined in the discussion section. Figure 2C shows the morphology of the two most common types of non-RPE that may arise during this differentiation process: neural or fibroblastic patches. It is important to note that these neural patches appear especially opaque on a dissecting microscope, whereas the defined, fibroblastic-like patches are nearly translucent on a dissecting microscope. It can be helpful to mark these areas on a tissue culture plate with an ethanol-proof lab pen to more easily identify them on both a compound light microscope and dissecting microscope. Figures 2D-F show the characteristic bright borders, cobblestone morphology, and pigmentation that indicate a healthy, maturing culture of RPE. Figure 3 is a higher magnification image to show the different appearance of fully mature RPE depicted by phase contrast and bright field microscopy. At passage 3 day 30, the cells are ready for the characterization that has been described in previous publications, including RNA expression, protein expression, growth factor secretion, and phagocytosis2,4,6,7. These characterizations show that the cells represented in these images are not only pigmented and cuboidal, but also phagocytic, post-mitotic, and polarized.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56274/56274fig1.jpg" /> 
Figure 1:. Timeline for the addition of growth factors and maturation of RPE. Growth factors are added to 12-well plates from day 0-14. Maturing RPE are cultured in 6-well plates or T75 flasks from day of enrichment to 30 days post-thaw (passage 3 day 30). Arrows indicate enzymatic cell passaging. (A-E) below the timelines correspond to the images in Figure 2. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56274/56274fig1large.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/56274/56274fig2.jpg" /> 
Figure 2: Representative morphology and confluence of maturing RPE. Induced pluripotent stem cells immediately before passaging for differentiation (A). Immature RPE cells subconfluent at day 2 (B) and before pick-to-remove enrichment on day 14; non-RPE patches (indicated by white arrows) appear as patches or opaque &#34;ribbons&#34; (C). RPE at passage 0, 1, and 3 on day 30 (D, E, and F). Scale bar = 200 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56274/56274fig2large.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/56274/56274fig3.jpg" /> 
Figure 3: Mature RPE at passage 3: Day 30 Cuboidal morphology depicted in phase contrast (A) and pigmentation depicted in bright-field (B). Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56274/56274fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells at JoVE.com

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

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

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

rapid-directed-differentiation-retinal-pigment-epithelial-cells-from

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.3K Views.  University of California, Santa Barbara. Dit protocol wordt beschreven hoe retinale pigment epitheliale cellen (RPE) uit pluripotente stamcellen te produceren. De methode gebruikt een combinatie van factoren die de groei en de kleine molecules om direct de differentiatie van stamcellen in onvolwassen RPE in veertien dagen en volwassen, functionele RPE na drie maanden.

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

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 ...

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

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

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

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

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

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.

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.

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

Genetics

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 ...

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

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

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

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

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Medicine

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 ...

Enhanced 3D Neural Retina Differentiation from Human Embryonic Stem Cells

Generating Neural Retina from Human Pluripotent Stem Cells

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of retinopathy. Unfortunately, ethical barriers hinder the collection of evidence from humans. Recently, numerous studies have shown that human pluripotent stem cells (PSCs) can be differentiated into retinal organoids (ROs) using different induction protocols, which have enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies. This study describes an optimized induction protocol to generate neural retina (NR) that significantly reduces the probability of vesiculation and fusion, increasing the success rate of production until day 60. Based on the ability of PSCs to self-reorganize after dissociation, combined with certain complementary factors, this new method can specifically drive NR differentiation. Furthermore, the approach is uncomplicated, cost-effective, exhibits notable repeatability and efficiency, presents encouraging prospects for personalized models of retinal diseases, and supplies a plentiful cell reservoir for applications such as cell therapy, drug screening, and gene therapy testing.

Author Spotlight: Simple and Efficient Neural Retina Organoid Production for Disease Modeling

The present protocol describes an optimized 3D neural retina induction system that reduces the adhesion and fusion of retinal organoids with high repeatability and efficiency.

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of ...

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.

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, ...

Biology

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...

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

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