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 border="0" cellpadding="0" cellspacing="0" width="722">
	<tbody>
		<tr height="36">
			<td height="36" style="height:36px;width:239px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:140px;">Catalog Number</td>
			<td style="width:161px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mTeSR1 media + 5X supplement</td>
			<td>Stem Cell Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632 (Rock Inhibitor)</td>
			<td>Stem Cell Technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:140px;">M-7154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Non essential amino acids</td>
			<td style="width:183px;">Hyclone(Fisher)</td>
			<td>SH30853.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Knockout serum replacement</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:140px;">10828-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gentamicin&#160;</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:140px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">L-Glutamine</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:140px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">MEM media</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:140px;">10370-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">N1 supplement</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:140px;">N-6530-5ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Taurine</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:140px;">T-8691-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Hydrocortisone</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:140px;">H0888-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Fetal bovine serum</td>
			<td style="width:183px;">Hyclone(Fisher)</td>
			<td>SH3008803HI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triiodo-l-thyronine sodium&#160; salt</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:140px;">T6397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Sodium hydroxide</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:140px;">S5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Dispase</td>
			<td style="width:183px;">Life Technologies</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Matrigel</td>
			<td style="width:183px;">BD Biosciences</td>
			<td style="width:140px;">354277</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Phosphate buffered saline</td>
			<td style="width:183px;">Hyclone(Fisher)</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Aggrewell 400 plate</td>
			<td style="width:183px;">Stem Cell Technologies</td>
			<td>27940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">AggreWell medium</td>
			<td>Stem Cell Technologies</td>
			<td>5893</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Accutase</td>
			<td>Stem Cell Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">BD Cytofix/Cytoperm Fixation/Permeabilization Kit</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Mouse Anti-PAX6 antibody</td>
			<td colspan="2">Developmental Studies Hybridoma Bank</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Rabbit Anti- RX antibody</td>
			<td>Abcam</td>
			<td>Ab23340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Mouse&#160; Anti-MITF antibody</td>
			<td>Thermo Scientific</td>
			<td>MS-772-P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Rabbit Anti-ZO-1 antibody</td>
			<td>Invitrogen</td>
			<td>40-2200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">RNeasy plus mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">PCR master mix</td>
			<td>promega</td>
			<td>M7502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">High capacity RNA to c DNA kit</td>
			<td style="width:183px;">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.  L'obiettivo di questa relazione è quello di descrivere i protocolli per derivare l'epitelio pigmentato retinico (RPE) da staminali pluripotenti indotte (iPS), le cellule con diverse dimensioni di corpi embrionali. 

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 ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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 ...

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 ...

Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent epiblast cells by orchestrated exposures to cytokines, including bone morphogenetic protein 4 (Bmp4). In human embryos, the earliest PGCs have been identified on the endodermal wall of yolk sac around the end of the 3rd week of gestation, but little is known about the process of human PGC specification and their early development. To circumvent the technical and ethical barriers of studying human embryonic PGCs, surrogate cell culture models have been recently generated from pluripotent stem cells. Here, we describe a 13-day protocol for robust production of human PGC-Like Cells (hPGCLCs). Human induced pluripotent stem cells (hiPSCs) maintained in the primed pluripotency state are incubated in the 4i na&#239;ve reprogramming medium for 48 hours, dissociated to single cells, and packed into microwells. Prolonged maintenance of hiPSCs in the na&#239;ve pluripotency state causes significant chromosomal aberrations and should be avoided. hiPSCs in the microwells are maintained for an additional 24 hours in the 4i medium to form embryoid bodies (EBs), which are then cultured in low-adherence plasticware under a rocking condition in the hPGCLC induction medium containing a high concentration of recombinant human BMP4. EBs are further cultured for up to 8 days in the rocking, non-adherent condition to obtain maximum yields of hPGCLCs. By immunohistochemistry, hPGCLCs are readily detected as cells strongly expressing OCT4 in almost all EBs exclusively on their surface. When EBs are enzymatically dissociated and subjected to FACS enrichment, hPGCLCs can be collected as CD38+ cells with up to 40-45% yield.

Primordial germ cells (PGCs) are common precursors of both sperm and eggs. Human embryonic PGCs are specified from pluripotent epiblast cells through interactions of cytokines. Here, we describe a 13-day protocol of inducing human cells transcriptomally resembling PGCs at the surface of embryoid bodies from primed-pluripotency induced pluripotent stem cells.

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...