The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. This research was performed while the authors Alberto Mu&#241;iz, Ramesh R Kaini, Whitney A Greene and Jae-Hyek Choi held a National Research Council Postdoctoral Research Associateship at the USAISR. This work was supported by U.S. Army Clinical Rehabilitative Medicine Research Program (CRMRP) and Military Operational Medicine Research Program (MOMRP).

1. Preparation of Culture Reagents and Culture Plates
<ol>
	<li>Prepare feeder-free stem cell culture medium by adding 100 ml of 5x serum-free supplement to 400 ml of stem cell basal medium. The medium is stable at 4 &#176;C for up to 2 weeks and at -20 &#176;C for 6 months.</li>
	<li>Add 10 &#181;M solution of rho-associated, coiled-coil containing protein kinase (Rock) inhibitor (Y-27362) to commercially available embryoid body (EB) formation medium.</li>
	<li>Prepare differentiation medium by adding 0.1 mM &#946;-mercaptoethanol, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 10% knockout serum replacement (KSR) and 10 &#181;g/ml gentamicin to Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12).</li>
	<li>Prepare iPS-RPE medium by adding 1x N1 supplement, 0.1 mM nonessential amino acids, 250 &#181;g/ml taurine, 13 ng/ml triiodo-L-thyronine sodium salt, 20 ng/ml hydrocortisone, 5 &#181;g/ml gentamicin, and 15% Fetal bovine serum (FBS) 21 to Minimum Essential Medium Eagle (MEM). Adjust the pH to 7.4.</li>
	<li>Prepare triiodo-L-thyronine sodium salt stock solution. Dissolve 1 mg of triiodo-L-thyronine sodium salt in 1 N sodium hydroxide by gently swirling. Add 49 ml of MEM to make 50 ml of 20 &#181;g/ml of triiodo-L-thyronine sodium salt. Prepare aliquots and freeze at -20 &#176;C until needed. Use appropriate volume to achieve desired concentration in final culture medium.</li>
	<li>Prepare hydrocortisone stock solution. Solubilize 1 mg of hydrocortisone in 1 ml of 100% ethanol by gentle agitation. Add 19 ml of MEM to make 20 ml of 50 &#181;g/ml hydrocortisone stock solution. Aliquot and freeze at -20 &#176;C until needed. Use appropriate volume to achieve the desired concentration in the final culture medium.</li>
	<li>Prepare a working solution of dispase of 1 mg/ml in DMEM/F12.</li>
	<li>Preparation of matrix coated plates
	<ol>
		<li>Prepare protein matrix extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells in DMEM/F12 to 0.08 mg/ml. Coat each well of a 6-well plate with 1 ml of the matrix solution. Incubate the coated plates at RT for 1 hr.</li>
		<li>After the 1 hr incubation, aspirate the excess DMEM/F12. Add 0.5 ml of feeder-free stem cell culture medium to prevent drying of the wells. The matrix coated plates are ready for use.</li>
	</ol>
	</li>
	<li>Prepare microwell plates, by rinsing each well with 2 ml of DMEM/F12. Remove DMEM/F12 and add 0.5 ml of EB formation medium supplemented with Rock inhibitor to each well. Centrifuge the plate at 2,000 x g for 5 min to remove any air bubbles.</li>
	<li>Prepare FACS staining buffer by mixing 2% FBS, and 0.09% sodium azide in phosphate buffered saline (PBS). Adjust the buffer to pH 7.4.</li>
	<li>Prepare trypsin neutralizing solution by adding 10% FBS to DMEM/F12.</li>
</ol>
2. iPS Culture
<ol>
	<li>Prior to iPS cell seeding, warm feeder-free stem cell culture medium to 37 &#176;C, and have the matrix coated plates ready.</li>
	<li>Quickly thaw the IMR90-1 iPS cells in a 37 &#176;C water bath. Then remove the cryo-vial from the water bath and wipe the vial with 70% ethanol. Transfer the cells to a 15 ml conical tube using a 2 ml pipette to minimize breaking of cell clumps.</li>
	<li>Add 5 ml of warm feeder-free stem cell culture medium to the cells dropwise and gently mix. Centrifuge the cells at 300 x g for 5 min at RT and remove the supernatant.</li>
	<li>Carefully resuspend the cell clumps in 2 ml of feeder-free stem cell culture medium and seed the cell clumps in the wells of matrix-coated plate. Place the plate into a 37 &#176;C incubator with 5% CO2, 95% humidity.</li>
	<li>Change the iPS cell culture medium daily. Observe undifferentiated colonies five to seven days after seeding. Check for undifferentiated colonies that are ready for passage (colonies with a dense center) under a light microscope.</li>
</ol>
3. Passaging of iPS Cells
<ol>
	<li>Prior to beginning to passage the iPS cells, warm the dispase solution, DMEM/F12 and feeder-free stem cell culture medium to 37 &#176;C in water bath.</li>
	<li>Before passaging iPS cells, remove any areas of differentiation by scraping the area and aspirating the medium. Carefully rinse the iPS cells with 2 ml DMEM/F12.</li>
	<li>Add 1 ml of 1 mg/ml dispase to each well and incubate at 37 &#176;C for 7 min. Aspirate the dispase and gently rinse the cell colonies with 2 ml of DMEM/F12 two times. After rinsing, add 2 ml of feeder-free stem cell culture medium to each well.</li>
	<li>Using a 5 ml pipette gently scrape the colonies of the plate to form cell clumps. Transfer the detached cell clumps to a 15 ml conical tube. Add sufficient feeder-free stem cell culture medium to seed the next passage of cells.</li>
</ol>
4. Generation of EBs Using Microwell Plates
<ol>
	<li>Preparation of a single cell suspension of iPS cells
	<ol>
		<li>Remove the medium from the iPS cells and rinse the iPS cells with 2 ml of DMEM/F12. Add 750 &#181;l of accutase to iPS cells at each well of the 6-well plate. Incubate the cells at 37 &#176;C and 5% CO2 to allow the cells to detach from the plate (approximately 5-10 min).</li>
		<li>Use a pipette to gently dissociate any remaining cells and to detach any remaining cells on the plate surface. Transfer the cells to a 50 ml conical tube. Rinse the plate with 5 ml of DMEM/F12 and combine the rinsed DMEM/F12 with the cells in the conical tube. Pass the cell suspension through a 40 &#181;m cell strainer to remove any possible residual cell clumps.</li>
		<li>Centrifuge the cells at 300 x g for 5 min at RT to remove the accutase. Resuspend the cell pellet in EB formation medium so that the cell concentration is approximately 0.5-1.0 x 107 cells/ml.</li>
		<li>Determine the number of viable cells by counting the cells with Trypan Blue and a hemocytometer.</li>
	</ol>
	</li>
	<li>Adjusting cell density in microwells to form size-controlled EBs
	<ol>
		<li>Adjust the number of cells to each well of the microwell plate to generate the desired EB sizes. Add cells to the microwells of the plates prepared in step 1.9, Evenly distribute the cells by gently pipetting the cells several times. 
		NOTE: Number of cells required per well = desired number of cells per EB x number of microwells per well.</li>
		<li>Adjust the EB formation medium supplemented with Rock inhibitor in the wells to a final volume of 2.0 ml. Redistribute the cells by gently pipetting each well. Centrifuge the microwell plates at 100 x g for 3 min. Place the plates in an incubator at 37 &#176;C with 5% CO2 and 95% humidity for 24 hr.</li>
	</ol>
	</li>
	<li>Harvesting EBs from the microwell plates
	<ol>
		<li>Harvest EBs from microwells by pipetting medium in the well up and down with a 1 ml micropipettor to remove most of the EBs. Pass the EB suspension through an inverted 40 &#181;m cell strainer on top of a 50 ml conical tube to remove single cells.</li>
		<li>Wash the microwell plate 5 times with 1 ml of DMEM/F12 to remove all the EBs. Collect washes and pass over the inverted cell strainer. Turn the cell strainer right-side up to a new 50 ml conical tube. Collect the EBs by washing with EB formation medium. Count EBs to determine the yield.</li>
	</ol>
	</li>
</ol>
5. Plating EBs and Initiating Differentiation
<ol>
	<li>Plate the EBs on protein matrix coated six well plates (as in step 1.8.1) at a density of &#60; 1,000 EBs/well in EB formation medium plus 10 &#181;M Rock inhibitor. Incubate the EBs at 37 &#176;C with 5% CO2 and 95% humidity for 24 hr.</li>
	<li>24 hr after EB plating, replace with differentiation medium to initiate differentiation. Change half of the differentiation medium every other day until the cells are collected for further analysis.</li>
	<li>Collect samples at day 6, 17, 29 and 60 to conduct RT-PCR, immunocytochemistry and FACS to validate expression of characteristic RPE genes and proteins.</li>
</ol>
6. RNA Extraction and PCR
<ol>
	<li>Extract RNA from the EB samples according to instructions provided in the commercially available kit. Determine RNA concentration by using a spectrophotometer.</li>
	<li>Perform reverse transcription on 100 ng of total RNA according to commercially available RNA to cDNA reverse transcript kit.</li>
	<li>Perform PCR with 10 ng of cDNA using the following primers (Table 1) at a concentration of 10 &#181;mol / 10 ng of cDNA. Set the PCR as follows: denature DNA at 95 &#176;C for 5 min, amplify with 35 cycles at 95 &#176;C for 15 sec, 60 &#176;C for 30 sec, 72 &#176;C for 1 min, followed by a final cycle at 72 &#176;C for 10 min. Run the PCR product on a 1% Agarose gel.</li>
</ol>
7. Immunocytochemistry
<ol>
	<li>Wash the EBs with PBS two times at RT. Fix the EBs at RT in 4% paraformaldehyde for 10 min. Rinse once with PBS at RT. Store samples in PBS at 4 &#176;C until used for staining.</li>
	<li>On staining day, treat the fixed cells with fixation and permeabilization reagents. Incubate the samples with blocking solution (10% goat serum in permeabilization solution) for 1 hr at RT.</li>
	<li>Perform immunochemistry in 10% goat serum in permeabilization solution using the following primary antibodies at the indicated dilutions:anti-Pax6 (1:10), anti-RX (1:200), anti-MITF (1:30), and anti-ZO1 (1:100). Incubate samples with corresponding antibodies O/N at 4 &#176;C.</li>
	<li>Next day, remove the primary antibody from the cells and rinse the samples three times with PBS. Incubate samples with fluorophore-conjugated secondary antibodies for 1 hr at RT.</li>
	<li>Remove the secondary antibody from the cells and rinse the samples three times in PBS. Mount the cover slips on glass slides with DAPI containing mounting medium and allow samples to set O/N in a dark chamber. Use a fluorescence microscope to visualize the staining.</li>
</ol>
8. Staining for FACS Analysis
<ol>
	<li>Wash the cultured EBs in PBS two times. Incubate the EBs with 0.5 ml of 0.25% trypsin for 5-10 min to make a single cell suspension. Neutralize the trypsin with trypsin neutralizing solution (1 ml/well).</li>
	<li>Transfer the single cell suspension to a 15 ml conical tube and centrifuge at 600 x g for 10 min.</li>
	<li>Discard the supernatant and add 10 ml of DMEM/F12 to the cells in the tube. Gently invert to mix the cells. Centrifuge at 600 x g. After centrifugation, discard the supernatant and resuspend the cells in FACS staining buffer.</li>
	<li>Count the total number of cells. Centrifuge at 600 x g for 10 min. After centrifugation discard supernatant. Fix the cells immediately by adding 0.5 ml of 4% paraformaldehyde. Mix well by gentle vortexing. Incubate the cells at 4 &#176;C for 20 min.</li>
	<li>Centrifuge at 600 x g for 10 min and remove the supernatant. Vortex gently to disrupt the cell pellet. Permeabilize the cells by adding 1 ml of chilled permeabilization solution. Vortex gently to mix and incubate on ice for 30 min.</li>
	<li>Centrifuge at 600 x g for 10 min and remove the supernatant. Add 3 ml of FACS staining buffer and resuspend. Repeat this step for one more time. Resuspend the cells in FACS staining buffer at a final concentration of 5 x 106 cells/ml.</li>
	<li>Transfer 100 &#956;l of the cell suspension (0.5 x 106 cells) to each microfuge tubes and add the recommended volume of primary antibody . For Pax6, add 5 &#181;l of PE anti-Pax6 in 100 &#181;l of cell suspension. For MITF, add 5 &#181;l of anti-MITF in 100 &#181;l of cell suspension.</li>
	<li>Mix and incubate at RT for 60 min. Add 3 ml of FACS staining buffer. Mix and centrifuge at 600 x g for 10 min. Remove the supernatant and repeat 8.16 for two additional times.</li>
	<li>For MITF staining, incubate the cells in secondary antibody, goat anti-mouse Alexa Fluor 488 at a dilution of 1:2,000 for 1 hr at 4 &#176;C. For Pax6 staining, avoid this step.</li>
	<li>Wash the cells with 5 ml of FACS staining buffer and centrifuge at 600 x g for 10 min. Repeat the process for two more times.</li>
	<li>Resuspend the cells in 500 &#956;l of FACS staining buffer and vortex gently to disrupt the cell pellet. Samples are ready for flow cytometric analysis.</li>
</ol>
9. iPS-RPE Isolation and Culture
<ol>
	<li>At day 29, carefully cut around the selected pigmented colonies from the culture plate using a 200 &#181;l pipette tip . Transfer the floating colonies to a 15 ml conical tube. Centrifuge the colonies at 600 x g for 10 min at RT and remove the supernatant.</li>
	<li>Resuspend the cell colonies in 10 ml of DMEM/F12 and centrifuge at 600 x g for 10 min. Remove the supernatant.</li>
	<li>Prepare a single cell suspension by incubating the colonies with 2 ml of 0.25% trypsin at 37 &#176;C for 7-10 min. Gently vortex the cell suspension to dissociate the colonies.</li>
	<li>Neutralize the trypsin by adding 2 ml of iPS-RPE medium. Centrifuge at 600 x g for 10 min and discard the supernatant. Resuspend the single cells in 2 ml of iPS-RPE medium. Transfer the cells into a protein matrix coated 6-well plate and place in an incubator at 37 &#176;C, 5% CO2 and 95% humidity.</li>
</ol>

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The authors have nothing to disclose.

To realize the full promise of pluripotent stem cells for cell therapy, it is necessary to regulate their differentiation in a consistent and reproducible way. This report describes protocols to form size-controlled EBs using microwell plate technology, initiate differentiation toward RPE and identify protein and gene markers of RPE. To synchronize the in vitro differentiation, homogenous sizes of EBs were formed by known numbers of iPS cells centrifuged in microwell plates by forced aggregation. Immunocytochemi...

Induced pluripotent stem (iPS) cells are a type of pluripotent stem cell derived by reprogramming adult cells with extrinsic factors1. In contrast, embryonic stem cells (ESCs), another type of pluripotent stem cell, are generated from the inner cell mass of the blastocyst2-3. Despite their different origins, iPS cells and ESCs are comparable in their unlimited capacity to replicate in vitro and in their capacity to differentiate into any cell type4-5. These characteristics of iPS cells make them ideal candidates for applications in personalized regenerative medicine. Recent research efforts are focused on developing robust differentiation protocols for producing specialized adult cells including retinal pigment epithelium (RPE)6-11.
For potential clinical applications of iPS derived cells, a directed differentiation for that specific cell type is essential. There are various methods published for directed differentiation of both ESCs and iPS cells into RPE that varies greatly in their efficiency6-7, 12-16. We still do not know many of the molecular events that govern the cell/tissue fate during development or differentiation. In recent years, efforts have been made to develop the differentiation protocol that can mimic the embryological development as much as possible. During the blastocyst phase, uncommitted population of stem cells are together in a three dimensional microenvironment. So, various strategies were applied to make the ESC/iPS cells assembled together and grow them in three dimensions. These stem cell aggregates are called embryoid bodies (EBs). Studies have shown that EB differentiation of stem cells mimic early stage of embryo development and can spontaneously give rise to primitive endoderm on its exterior surface. Later, as EB development progresses, differentiated cell phenotypes of all three germ lineages appear17-18. Therefore, EBs based differentiation protocols have attracted a lot of attention for in vitro differentiation of ESC/iPS cells and are a good candidate for RPE generation from pluripotent stem cells13.
EBs can be made using several methods from ESC/iPS cells. Initially, EBs were made by scraping off adherent colonies and maintaining them in non-adherent suspension culture. However, this approach yields heterogeneous population of EBs that causes low reproducibility. Hanging drop cell culture and microwell based EBs formation are other popular techniques for EBs formation which yield homogenous EBs of defined sizes that are highly reproducible. Furthermore, the microwell technique can yield large number of aggregates with less effort.
Differentiation of cells within EBs is regulated by a multiplex of morphogenic cues from the extracellular and intracellular microenvironment. In contrast to differentiation in a monolayer format, EBs provide a platform for complex assembly of cells and intercellular signaling to occur17. Interestingly, the number of pluripotent stem cells used to make individual EBs was observed to influence the fate of cells. For example, in a hematopoietic differentiation study of human ESCs, it was observed that 500-cell EB promoted differentiation towards myeloid lineage whereas 1,000-cell EB pushed towards erythroid lineage20. In another study, smaller EBs favored endoderm differentiation whereas larger EBs promoted towards neuro-ectoderm differentiation11, 17.
These past studies strongly suggest that the number of ESC/iPS cells used to make individual EBs affect the EBs based differentiation to any cell types. However, to our knowledge, there are no current studies that have elucidated the impact of EBs size in its propensity to differentiate towards RPE. The goal of this study is to characterize the influence of EB size on induced pluripotent stem (iPS) cells - retinal pigment epithelium (iPS-RPE) differentiation and to identify the optimum cell number to make the EBs for directed differentiation towards RPE lineage.

<table><tbody><tr><td>mTeSR1 media + 5x supplement</td><td>Stem Cell Technologies</td><td>5850</td><td></td></tr><tr><td>Y-27632 (Rock Inhibitor)</td><td>Stem Cell Technologies</td><td>72304</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>11330-032</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M-7154</td><td></td></tr><tr><td>Non essential amino acids</td><td>Hyclone(Fisher)</td><td>SH30853.01</td><td></td></tr><tr><td>Knockout serum replacement</td><td>Life Technologies</td><td>10828-028</td><td></td></tr><tr><td>Gentamicin&amp;nbsp;</td><td>Life Technologies</td><td>15750-060</td><td></td></tr><tr><td>L-Glutamine</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>MEM media</td><td>Life Technologies</td><td>10370-021</td><td></td></tr><tr><td>N1 supplement</td><td>Sigma</td><td>N-6530-5ML</td><td></td></tr><tr><td>Taurine</td><td>Sigma</td><td>T-8691-25G</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma</td><td>H0888-1G</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Hyclone(Fisher)</td><td>SH3008803HI</td><td></td></tr><tr><td>Triiodo-L-thyronine sodium&amp;nbsp; salt</td><td>Sigma</td><td>T6397</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>S5881</td><td></td></tr><tr><td>Dispase</td><td>Life Technologies</td><td>17105-041</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354277</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Hyclone(Fisher)</td><td>10010-023</td><td></td></tr><tr><td>Aggrewell 400 plate</td><td>Stem Cell Technologies</td><td>27940</td><td></td></tr><tr><td>AggreWell medium</td><td>Stem Cell Technologies</td><td>5893</td><td></td></tr><tr><td>Accutase</td><td>Stem Cell Technologies</td><td>7920</td><td></td></tr><tr><td>BD Cytofix/Cytoperm Fixation/Permeabilization Kit</td><td>BD Biosciences</td><td>554714</td><td></td></tr><tr><td>Mouse Anti-PAX6 antibody</td><td>Developmental Studies Hybridoma Bank</td><td></td><td></td></tr><tr><td>Rabbit Anti-RX antibody</td><td>Abcam</td><td>Ab23340</td><td></td></tr><tr><td>Mouse&amp;nbsp; Anti-MITF antibody</td><td>Thermo Scientific</td><td>MS-772-P</td><td></td></tr><tr><td>Rabbit Anti-ZO-1 antibody</td><td>Invitrogen</td><td>40-2200</td><td></td></tr><tr><td>RNeasy plus mini kit</td><td>Qiagen</td><td>74134</td><td></td></tr><tr><td>PCR master mix</td><td>Promega</td><td>M7502</td><td></td></tr><tr><td>High capacity RNA to cDNA kit</td><td>Life Technologies</td><td>4387406</td><td></td></tr></tbody></table>

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.

In this experiment, iPS cells were cultured and differentiated into the RPE lineage from EBs. EBs of controlled sizes were formed using microwell plates. As seen in Figure 1 EB formation was homogenous in the microwell plates. These EBs were then collected and plated on 6-well plates (Figure 2).
RPE can be identified by their classical hexagonal morphology, pigmentation, and expression of RPE markers. After 12 weeks of culture, the 200-cell EBs had developed a...

Watch this Scientific Journal Video about Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies at JoVE.com

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.

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 ...

deriving-retinal-pigment-epithelium-rpe-from-induced-pluripotent-stem

Research

JoVE Journal

Developmental Biology

10.5K Views.  U.S. Army Institute of Surgical Research.  Het doel van dit rapport is om de protocollen beschrijven aan de netvliespigmentepitheel (RPE) van geïnduceerde pluripotente stamcellen (iPS) cellen met behulp van verschillende maten van embryoid lichamen af ​​te leiden. 

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

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 ...

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 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 ...

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 ...

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

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

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

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

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

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