Name: Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies (Video) | JoVE Journal
Uploaded: 2015-02-04T14:00:00.000+00:00
Duration: 9 min 18 s
Description: Learn to derive retinal pigment epithelium from induced pluripotent stem cells using controlled embryoid body sizes. This microwell technique optimizes RPE differentiation efficiency for retinal disease research.

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5 minutes

The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. This research was performed while the authors 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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<li>Yoshida, Y., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((iPS+cells%3A+a+source+of+cardiac+regeneration%5BTitle%5D)+AND+%22J+Mol+Cell+Cardiol%22%5BJournal%5D)">iPS cells: a source of cardiac regeneration.</a> J Mol Cell Cardiol. 50 (2), 327-332 (2011).</li>
<li>Mak, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+molecules+greatly+improve+conversion+of+human-induced+pluripotent+stem+cells+to+the+neuronal+lineage%5BTitle%5D)+AND+%22Stem+Cells+Int%22%5BJournal%5D)">Small molecules greatly improve conversion of human-induced pluripotent stem cells to the neuronal lineage.</a> Stem Cells Int. 2012, 140427(2012).</li>
<li>Bauwens, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+human+embryonic+stem+cell+colony+and+aggregate+size+heterogeneity+influences+differentiation+trajectories%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories.</a> Stem Cells. 26 (9), 2300-2310 (2008).</li>
<li>Cour la, M., Tezel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Retinal+Pigment+Epithelium%5BTitle%5D)+AND+%22Advances+in+Organ+Biology%22%5BJournal%5D)">The Retinal Pigment Epithelium.</a> Advances in Organ Biology. 10, 253-273 (2006).</li>
<li>Vaajassari, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toward+the+defined+and+xeno-free+differentiation+of+functional+human+pluripotent+stem+cell-derived+retinal+pigment+epithelial+cells%5BTitle%5D)+AND+%22Mol+Vis%22%5BJournal%5D)">Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells.</a> Mol Vis. 17, 558-575 (2011).</li>
<li>Carr, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protective+effects+of+human+iPS-derived+retinal+pigment+epithelium+cell+transplantation+in+the+retinal+dystrophic+rat%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat.</a> PLoS One. 4 (12), e8152(2009).</li>
<li>Muniz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinoid+uptake%2C+processing%2C+and+secretion+in+human+iPS-RPE+support+the+visual+cycle%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">Retinoid uptake, processing, and secretion in human iPS-RPE support the visual cycle.</a> Invest Ophthalmol Vis Sci. 55 (1), 198-209 (2014).</li>
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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

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...

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 ...

The overall goal of this procedure is to characterize the influence of embryo body size on RPE differentiation from IPS cells for directing embryo body differentiation towards the RPE lineage. This is accomplished by first using the microwell technique to generate homogeneous embryo bodies of defined sizes. In the second step directed differentiation of the IPS cells is initiated towards the retinal lineage, and then the I-P-S-R-P-E cells are identified and isolated.In the final step, the I-P-S-R-P-E cells are enriched and expanded. Ultimately, the I-P-S-R-P-E cells can be analyzed for their expression of I-P-S-R-P-E specific markers. The main advantage of this technique over existing methods like spontaneous differentiation, is that our technique results in higher of RP.This matter can help to optimize the directed differentiation of IPS cells towards the RP lineage.The implications of this technique extend toward the therapy of retinal degenerative diseases such as macular degeneration, retinitis figment, or stargardt's disease. Since the replacement of diseased RPE with healthy RPE derived from IPS cells may be the best option for preventing loss of vision, generally Individual new to this method will struggle because stem cells are very delicate and subtle changes in their handle can affect the differentiation process. Visual demonstration of this method is critical as the embryo body harvesting step from the microwave place is difficult.Prior to seeding warm the feeder free stem cell culture medium to 37 degrees Celsius, and then quickly warm the IMR 90 dash one IPS cells in a 37 degree Celsius water bath. When the cells are thawed, add five milliliters of warm feeder free stem cell culture, medium to the cells, dropwise and gently mix. Next, spin down the cells and then carefully resuspend the cell clumps in two milliliters of feeder free stem cell culture.Medium seed the clumps in individual wells of a matrix coated plate, and then place the cells in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide and 95%humidity and daily medium changes. Five to seven days later, observe the undifferentiated colonies with a dense center that are ready for passage under a light microscope. When the cells are ready, add one milligram per milliliter of 37 degrees Celsius warm dis space to each well and incubate the plate at 37 degrees Celsius for seven minutes.Then aspirate the DYS space and gently rinse the cell colonies with two milliliters of prewarm DM MF 12. Two times. After rinsing, add two milliliters of feeder free stem cell culture medium to each well, and use a five milliliter pipette to gently scrape the colonies, releasing the cell clumps.Then transfer the detached clumps into a 15 milliliter conical tube and add sufficient feeder free stem cell culture medium to seed the next passage of cells to generate the embryo bodies. First, rinse the IPS cells with two milliliters of DMMF 12 and then add 750 microliters of Accutane to each well of the six well plate for five to 10 minutes. In a cell culture incubator, when the cells have begun to detach, use a pipette to gently dissociate the cells into a single cell suspension and then transfer them into a 50 milliliter conical tube.Rinse the plate with five milliliters of DM MF 12, pooling the wash with the cells in the conical tube. Then pass the cell suspension through a 40 micrometer cell strainer to remove any residual cell clumps. Now spin down the cells to remove the Accutane and resus.Suspend the pellet in embryo body formation medium to a 0.5 to one times 10 to the seventh cells per milliliter concentration. After determining the number of viable cells by trian blue exclusion, add the appropriate number of cells to each well of a micro well plate to generate the desired embryo. Body sizes.Gently pipette the cultures to facilitate an even distribution of the cells, and then adjust the embryo body formation medium supplemented with rock inhibitor to a final volume of 2.0 milliliters. After more gentle pipetting, spin down the micro well cell cultures and incubate the plate in a cell culture incubator for 24 hours. The next day pipette medium up and down into each micro well with a one milliliter micro pipetter to harvest most of the embryo bodies and then pass the embryo body suspension through an inverted 40 micrometer cell strainer to remove any single cells.Next, rinse the micro well plate five times with one milliliter of DMMF 12 to remove the rest of the embryo bodies passing the washes through the strainer. Then turn the cell strainer upside down in a Petri dish and filter the collected embryo bodies with more embryo body formation medium. After counting the collected cells incubate the embryo bodies on protein matrix coated six well plates at less than 1000 embryo bodies per well in embryo body formation medium, supplemented with 10 micromolar rock inhibitor in a cell culture incubator for 24 hours.Finally, replace the embryo body formation medium with differentiation medium to initiate the differentiation. Collecting samples at day 6, 17, 29 and 60 to conduct the appropriate downstream analysis. After 12 weeks of culture, 200 cell embryo bodies develop astrocyte and fibroblast morphology with no visible pigmentation, whereas the larger embryo bodies develop a monolayer of classical RPE morphology and pigmentation as demonstrated in these images immunochemistry to detect the RPE markers, MITF and ZO one reveals the co-expression of these proteins derived from 500 cell and 3000 cell embryo bodies.In this graph, the neuroectodermal marker PAC six was measured in differently sized embryo bodies at different time points by flow cytometry. For example, in the 3000 cell embryo bodies, approximately 50%of the analyzed cells were positive for PAC six on day six of culture. Additionally, fax analysis of the RPE marker MITF on embryo bodies of various sizes revealed that 20%of the cells expressed MITF by day 60 of differentiation.Cultured RPE are also characterized by their ability to lose their pigment and polygonal morphology and to obtain a fibroblast phenotype upon packaging. For example, here, newly passage cells that have lost their pigment and have gained a fibroblast morphology are shown. These cells then proliferate, regaining their classical polygonal morphology upon confluence, and within a few weeks, regain their pigmentation.While attempting this procedure, it's important to remember to properly identify and isolate the IPS RP colonies After its development. This technique paved the way for researchers in the field of stem cell biology to explore the directed differentiation towards many adult cell types by mimicking normal embryological development. Don't forget that working in the tissue, virtual labs can be extremely hazardous, and that repercussions, such as wearing leg coats, gloves, and glasses should always be taken while doing these procedures.After watching this video, you should have a good understanding of how to generate homogeneous embryo bodies of the desire size to direct the deforestation towards I-P-S-R-P-E and to isolate and enrich RPE cells.

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

Research

JoVE Journal

Developmental Biology

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.

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

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

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

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

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

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

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

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

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

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

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.

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

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

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

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

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

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

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

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.

Generating Neural Retina from Human Pluripotent Stem Cells

Learn to derive retinal pigment epithelium from induced pluripotent stem cells using controlled embryoid body sizes. This microwell technique optimizes RPE differentiation efficiency for retinal disease research.

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

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