Name: engineering transplantation-suitable retinal pigment epithelium tissue derived from human embryonic stem cells
Uploaded: 2018-09-06T15:00:00
Description: Watch this Scientific Journal Video about Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells at JoVE.com

The authors would like to thank J&#233;r&#244;me Larghero and Val&#233;rie Vanneaux (H&#244;pital Saint Louis, Paris, France) for their input during the setting-up of the method described here.
This work was supported by grants from the ANR [GPiPS: ANR-2010-RFCS005; SightREPAIR: ANR-16-CE17-008-02], the Fondation pour la Recherche M&#233;dicale [Bio-engineering program - DBS20140930777] and from LABEX REVIVE [ANR-10-LABX-73] to Olivier Goureau and Christelle Monville. It was supported by NeurATRIS, a translational research infrastructure (Investissements d&#39;Avenir) for biotherapies in Neurosciences [ANR-11-INBS-0011] and INGESTEM, the national infrastructure (Investissements d&#39;Avenir) engineering for pluripotent and differentiated stem cells [ANR-11-INBS-000] to Christelle Monville. Karim Ben M&#39;Barek was supported by fellowships from DIM Stempole and LABEX REVIVE [ANR-10-LABX-73]. I-Stem is part of the Biotherapies Institute for Rare Diseases supported by the Association Fran&#231;aise contre les Myopathies (AFM)-T&#233;l&#233;thon.

All human materials used in this protocol were used in accordance with European Union regulations. The human ES cell line used in this study was derived from a unique embryo. The couple who had donated the embryo was fully informed and gave their consent for an anonymous donation. A clinical-grade human ES cell line was derived from this embryo, banked, qualified, and properly documented by Roslin Cells (UK). hAMs were procured under sterile conditions during a cesarean section in mothers who signed an informed consent f...

<ol>
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Global Health. 2 (2), e106-e116 (2014).</li><li>Ben M'Barek, K., Regent, F., Monville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+human+pluripotent+stem+cells+to+study+and+treat+retinopathies.">Use of human pluripotent stem cells to study and treat retinopathies.</a> World Journal of Stem Cells. 7 (3), 596-604 (2015).</li><li>Ben M'Barek, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+ESC-derived+retinal+epithelial+cell+sheets+potentiate+rescue+of+photoreceptor+cell+loss+in+rats+with+retinal+degeneration.">Human ESC-derived retinal epithelial cell sheets potentiate rescue of photoreceptor cell loss in rats with retinal degeneration.</a> Science Translational Medicine. 9 (421), (2017).</li><li>Schwartz, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+trials+for+macular+degeneration:+a+preliminary+report.">Embryonic stem cell trials for macular degeneration: a preliminary report.</a> Lancet. 379 (9817), 713-720 (2012).</li><li>Schwartz, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+retinal+pigment+epithelium+in+patients+with+age-related+macular+degeneration+and+Stargardt's+macular+dystrophy:+Follow-up+of+two+open-label+phase+1/2+studies.">Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt's macular dystrophy: Follow-up of two open-label phase 1/2 studies.</a> Lancet. 385 (9967), 509-516 (2015).</li><li>Hsiung, J., Zhu, D., Hinton, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+human+embryonic+stem+cell-derived+retinal+pigment+epithelial+cell+monolayers+have+higher+resistance+to+oxidative+stress-induced+cell+death+than+nonpolarized+cultures.">Polarized human embryonic stem cell-derived retinal pigment epithelial cell monolayers have higher resistance to oxidative stress-induced cell death than nonpolarized cultures.</a> Stem Cells Translational Medicine. 4 (1), 10-20 (2015).</li><li>Diniz, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+implantation+of+retinal+pigment+epithelial+cells+derived+from+human+embryonic+stem+cells:+improved+survival+when+implanted+as+a+monolayer.">Subretinal implantation of retinal pigment epithelial cells derived from human embryonic stem cells: improved survival when implanted as a monolayer.</a> Investigative Ophthalmology &amp; Visual Science. 54 (7), 5087-5096 (2013).</li><li>Kamao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+induced+pluripotent+stem+cell-derived+retinal+pigment+epithelium+cell+sheets+aiming+for+clinical+application.">Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application.</a> Stem Cell Reports. 2 (2), 205-218 (2014).</li><li>Mandai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+induced+stem-cell-derived+retinal+cells+for+macular+degeneration.">Autologous induced stem-cell-derived retinal cells for macular degeneration.</a> The New England Journal of Medicine. 376 (11), 1038-1046 (2017).</li><li>Thomas, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+functionality+of+hESC-derived+retinal+pigment+epithelium+cells+cultured+as+a+monolayer+on+polymer+substrates+transplanted+in+RCS+rats.">Survival and functionality of hESC-derived retinal pigment epithelium cells cultured as a monolayer on polymer substrates transplanted in RCS rats.</a> Investigative Ophthalmology &amp; Visual Science. 57 (6), 2877-2887 (2016).</li><li>Borooah, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+human+induced+pluripotent+stem+cells+to+treat+retinal+disease.">Using human induced pluripotent stem cells to treat retinal disease.</a> Progress in Retinal and Eye Research. 37, 163-181 (2013).</li><li>Leach, L. L., Clegg, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+Making+stem+cells+retinal:+Methods+for+deriving+retinal+pigment+epithelium+and+implications+for+patients+with+ocular+disease.">Concise review: Making stem cells retinal: Methods for deriving retinal pigment epithelium and implications for patients with ocular disease.</a> Stem Cells. 33 (8), 2363-2373 (2015).</li><li>Reichman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+confluent+human+iPS+cells+to+self-forming+neural+retina+and+retinal+pigmented+epithelium.">From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (23), 8518-8523 (2014).</li><li>Lustremant, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cells+as+a+tool+to+model+a+form+of+Leber+congenital+amaurosis.">Human induced pluripotent stem cells as a tool to model a form of Leber congenital amaurosis.</a> Cellular Reprogramming. 15 (3), 233-246 (2013).</li><li>Reichman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+storable+retinal+organoids+and+retinal+pigmented+epithelium+from+adherent+human+iPS+Cells+in+xeno-free+and+feeder-free+conditions.">Generation of storable retinal organoids and retinal pigmented epithelium from adherent human iPS Cells in xeno-free and feeder-free conditions.</a> Stem Cells. 35 (5), 1176-1188 (2017).</li><li>Maruotti, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule-directed,+efficient+generation+of+retinal+pigment+epithelium+from+human+pluripotent+stem+cells.">Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (35), 10950-10955 (2015).</li><li>Stanzel, B. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+RPE+stem+cells+grown+into+polarized+RPE+monolayers+on+a+polyester+matrix+are+maintained+after+grafting+into+rabbit+subretinal+space.">Human RPE stem cells grown into polarized RPE monolayers on a polyester matrix are maintained after grafting into rabbit subretinal space.</a> Stem Cell Reports. 2 (1), 64-77 (2014).</li><li>Ilmarinen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin+polyimide+membrane+as+cell+carrier+for+subretinal+transplantation+of+human+embryonic+stem+cell+derived+retinal+pigment+epithelium.">Ultrathin polyimide membrane as cell carrier for subretinal transplantation of human embryonic stem cell derived retinal pigment epithelium.</a> PloS One. 10 (11), e0143669 (2015).</li><li>Thumann, G., Schraermeyer, U., Bartz-Schmidt, K. U., Heimann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet's+membrane+as+membranous+support+in+RPE/IPE+transplantation.">Descemet's membrane as membranous support in RPE/IPE transplantation.</a> Current Eye Research. 16 (12), 1236-1238 (1997).</li><li>Kiilgaard, J. F., Scherfig, E., Prause, J. 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O., Clegg, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cell-based+therapies+in+combination+with+substrate+for+the+treatment+of+age-related+macular+degeneration.">Pluripotent stem cell-based therapies in combination with substrate for the treatment of age-related macular degeneration.</a> Journal of Ocular Pharmacology and Therapeutics: The Official Journal of the Association. 32 (5), 261-271 (2016).</li><li>Song, M. J., Bharti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+into+the+future:+Using+induced+pluripotent+stem+cells+to+build+two+and+three+dimensional+ocular+tissue+for+cell+therapy+and+disease+modeling.">Looking into the future: Using induced pluripotent stem cells to build two and three dimensional ocular tissue for cell therapy and disease modeling.</a> Brain Research. 1638 (Pt A), 2-14 (2016).</li><li>Ramsden, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+retinal+regeneration:+Past,+present+and+future).">Stem cells in retinal regeneration: Past, present and future).</a> Development. 140 (12), 2576-2585 (2013).</li><li>da Cruz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+1+clinical+study+of+an+embryonic+stem+cell-derived+retinal+pigment+epithelium+patch+in+age-related+macular+degeneration.">Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration.</a> Nature Biotechnology. 36 (4), 328-337 (2018).</li><li>Kashani, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioengineered+retinal+pigment+epithelial+monolayer+for+advanced,+dry+age-related+macular+degeneration.">A bioengineered retinal pigment epithelial monolayer for advanced, dry age-related macular degeneration.</a> Science Translational Medicine. 10 (435), (2018).</li><li>Binder, S., Stanzel, B. V., Krebs, I., Glittenberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+the+RPE+in+AMD.">Transplantation of the RPE in AMD.</a> Progress in Retinal and Eye Research. 26 (5), 516-554 (2007).</li><li>Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., Hjelmeland, L. 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We described a method for the culture of RPE cells on a biological scaffold and its preparation for implantation in animal models. One of the critical steps of the protocol is the maintenance of the orientation of the hAM all along the procedure until its inclusion into gelatin. Indeed, the native epithelium of the membrane is removed and its basement membrane becomes exposed9. The RPE cells have to be seeded on top of this basement membrane. Upon preparation for gelatin embedding, it is crucial t...

RPE is crucial for the survival and homeostasis of the photoreceptors with which it is tightly associated1. Several pathological conditions alter its functionality and/or survival, including RP and AMD.
RP is a group of inherited monogenic mutations that affect the functions of photoreceptors or RPE cells or both2,3. It is estimated that mutations that affect specifically the RPE cells account for 5% of RP2. AMD is another condition where the RPE layer is altered, leading ultimately to central vision loss. AMD is caused by the complex interactions of genetic and environmental factors and affects the elderly4,5,6. According to projections, AMD will be a concern for 196 million patients worldwide by 20207. For these disorders, no effective cure exists, and one of the strategies proposed is the transplantation of new RPE cells in order to compensate for dead/nonfunctional preexisting RPE cells8.
The mode of formulation of the final product to be grafted is essential to ensure the best functional outcomes. RPE cells injected as a cell suspension, despite being an easy and straightforward method of delivery, raise concerns regarding their survival, integration, and functionality9,10,11,12,13. Scientists are now developing more complex formulations to deliver engineered retinal tissue9,13,14,15,16. In this context, we developed an original method to generate in vitro RPE tissue that could be used for transplantation9.
RPE cell banks derived from human embryonic stem (ES) cells are used in this protocol. However, alternative RPE cell banks from different cell sources (human-induced pluripotent stem cells, primary RPE cells, etc.) and differentiated with a different method are also suitable for this protocol. It includes directed differentiation protocols using cytokines and/or small molecules17,18,19,20,21,22.
To be transplanted, the engineered tissue should be prepared on a scaffold. In the past few years, different scaffolds were developed based on a polymer or on a matrix of biological origin13,23,24. Here, the biological substrate used is the hAM, but other substrates, like denuded Bruch membranes, could be implemented. The method described herein has the advantage of using a biological scaffold that is more relevant to the RPE native environment.
Human ES cell-derived RPE cells are cultured for at least 4 weeks in order to be fully organized as a cobblestone monolayer. At that stage, the epithelium obtained is functional and polarized9. Finally, as this tissue wrinkles easily, it is embedded in a thin layer of a hydrogel carrier to give it more rigidity and elasticity and to protect it during the injection procedure. This product is then stored at 4 &#176;C until grafting.

<table>
	<tbody>
		<tr>
			<td>Sterile biosafety cabinet</td>
			<td>TechGen International</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid waste disposal system for aspiration</td>
			<td>Vacuubrand</td>
			<td>BVC 21</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2-controlled +37&#160;&#176;C cell incubator</td>
			<td>Thermo Electron Corporation</td>
			<td>BVC 21 NT</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#160;&#181;L pipette: P200</td>
			<td>Gilson</td>
			<td>F144565</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#160;mL pipette: P1000</td>
			<td>Gilson</td>
			<td>F144566</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet aid</td>
			<td>Drummond</td>
			<td>75001</td>
			<td></td>
		</tr>
		<tr>
			<td>+4&#160;&#176;C refrigerator</td>
			<td>Liebherr</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>WPI</td>
			<td>501758</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (x2)</td>
			<td>WPI</td>
			<td>555227F</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Grant subaqua pro</td>
			<td>SUB6</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision balance</td>
			<td>Sartorius</td>
			<td>CP225D</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorff</td>
			<td>5804</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>SC30</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal Rocking Shaker</td>
			<td>IKA-WERKE</td>
			<td>IKA MTS 214D</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR</td>
			<td>LAB DANCER S40</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel</td>
			<td>WPI</td>
			<td>500351</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic paraffin film</td>
			<td>VWR</td>
			<td>PM992</td>
			<td></td>
		</tr>
		<tr>
			<td>0.200 &#181;m single use syringe filter</td>
			<td>SARTORIUS</td>
			<td>16532</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe without needle 50 mL</td>
			<td>Dutscher</td>
			<td>50012</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottles 250mL</td>
			<td>Dutscher</td>
			<td>28024</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile Falcon tubes</td>
			<td>Dutscher</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile Falcon tubes</td>
			<td>Dutscher</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>culture insert</td>
			<td>Scaffdex</td>
			<td>C00001N</td>
			<td></td>
		</tr>
		<tr>
			<td>60&#160;mm cell culture disches: B6</td>
			<td>Dutscher</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well cell culture plate</td>
			<td>Corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Ted Pella, Inc</td>
			<td>121-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>Castorama</td>
			<td>3178040670105</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X (500&#160;mL)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermolysine</td>
			<td>Roche</td>
			<td>5339880001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX</td>
			<td>Invitrogen</td>
			<td>61965-026</td>
			<td></td>
		</tr>
		<tr>
			<td>KSR CTS (KnockOut SR XenoFree CTS)</td>
			<td>Invitrogen</td>
			<td>12618-013</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM-NEAA (100X)</td>
			<td>Invitrogen</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>b-mercaptoethanol (50&#160;mM)</td>
			<td>Invitrogen</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2-independent medium</td>
			<td>GIBCO</td>
			<td>18045-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>MERCK</td>
			<td>104078</td>
			<td></td>
		</tr>
		<tr>
			<td>human amniotic membrane</td>
			<td>Tissue bank St Louis hospital (Paris, France)</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

hAMs contain an epithelial layer that should be removed before the seeding of RPE cells. An enzymatic treatment of the membrane is performed with the thermolysin under shaking. In order not to not lose the polarity of the membrane (the epithelium is on one side), it is fixed on a support which composition could be different depending on the provider (Figure 1A). Check the adhesion of the membrane to its support at this step and add clips if necessary. At the ...

Watch this Scientific Journal Video about Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells at JoVE.com

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

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 ...

This method can help answer key questions about the self therapy of the eye, such as what is the efficacy of a specifically designed bio-engineered tissue in vivo. With this technique we provide a method of producing and contouring the retinal tissue as well as a strategy for processing the tissue for eye implantation in an animal model. Begin by washing one to four 30 by 30 mm pieces of human amniotic membrane, or hAM.Fix a nylon scaffolds two times in 80ml of PBS per wash in a 250ml bottle on a shaker, at high speed for five minutes. After the second wash, add 40ml of working Thermolysin solution to each bottle, and shake the bottles two times for five minutes at 450 rpm, followed by 30 seconds of vortexing. After the second round of Thermolysin treatment, wash the pieces two more times with 80ml of PBS per wash, as demonstrated.Then, use long sterile forceps to transfer the hAMs individually, into a 10cm culture dish, containing 10ml of PBS. Next place one membrane with the nylon facing down in a second 10cm culture dish of PBS, and detach two of the four clips holding the membrane to nylon. Insert the smaller ring of the culture insert between the nylon and the membrane, making sure that the membrane covers the smaller ring completely.Clip the second part of the culture insert to the top of the membrane so that the basement membrane of the hAM is facing right side up in the culture insert and detach the last two clips. Then use sterile scissors to trim any membrane outside of the culture insert as necessary. And use forceps to transfer the membrane into one well of a 12 well plate, containing PBS for storage at 37 degrees Celsius and 5%CO2.Before seeding, warm cryo vials containing one times 10 to the sixth RPE cells in 3ml of RPE medium in one 15ml conical tube per vial. When the cells have thawed, pipette to obtain single cell suspensions in each of the tubes, and count the number of viable cells by tri pan blue exclusion. Collect the cells by centrifugation and re suspend the pellets at seven times 10 to the fifth cells per milliliter concentration in fresh RPE medium.Next, aspirate the PBS from each well of the prepared 12 well plate, and add 500 micro liters of cells to the center of each culture insert, and 1ml of RPE medium to the bottom of each well. Then return the plate to the cell culture incubator for 30 days, changing the medium two times per week until the end of the culture. On culture day 30, warm an 8%gelatin solution in a 37 degrees Celsius water bath, for 30 minutes.And fill the internal chamber of a vibratome with four degrees Celsius CO2 independent medium. Use a scalpel to cut a 5cm by 5cm block from a 20%gelatin block stored at four degrees Celsius. Secure the top side of the block to the support with an appropriate adhesive.And place a new blade into the vibratome. Adjust the block until it is at the same level as the razor blade, and fill the bath around the support with fresh four degrees Celsius CO2 independent medium. Cut the block at a medium velocity until the block is cut uniformly with a smooth leading surface and set the position to zero.Then aspirate the medium around the gelatin until the gelatin is completely dry. And use forceps to carefully open an RPE seeded culture insert on top of the block. Use scissors to remove all of the membrane outside of the insert ring, and aspirate the entire volume of residual medium.Add up to 1ml of 8%liquid gelatin solution to the membrane until it is covered. And carefully remove any excess gelatin. After five to eight minutes, add fresh four degrees Celsius CO2 independent medium to the bath until the solidified membrane is covered.And with the vibratome at the 100 micron position, cut a section of the membrane at a medium velocity, taking care to maintain the orientation of the tissue. Cut a corner of the section with a scalpel to allow identification of the orientation of the section. And use a spatula to transfer the gelatin-embedded membrane to one of a six well plate, filled with four degrees Celsius conservation medium.The orientation of the slice could be inverted due to freed movement before the corner being cut. So take care to secure the position of the slice just after the section. The size of the implant can then be adjusted to the size of the recipient eye under a surgical microscope, prior transplantation.Typically a few dead cells remain on the surface of the membrane, after fixation on the culture insert, but those cells are eliminated when the culture medium is changed. The fibers of the basement membrane are visible at a higher magnification when no cells remain, indicating that the Thermolysin treatment period has been optimized. The retinal epithelium becomes easier to see under a microscope, as it matures, forming a complete cobblestone-like monolayer at three weeks.At four weeks, the epithelium is mature enough to be prepared for implantation. While attempting this procedure, it's important to remember that the state of the gelatin is dependent on the temperature. So take care to strictly follow recommended temperatures.After its development, this technique paves the way for researchers in the field of stem cell therapy, to explore the therapeutic potential of a bio-engineered tissue in an animal model of retinitis pigmentosa. Though this method can be used to prepare an tissue on top of a scaffold, for its subsequent transplantation, it can also be applied to more complex systems containing photo receptor layers. Generally, individuals new to this method will struggle because of the difficulty in handling the human amniotic membranes, which is very thin and tends to fold easily.Visual demonstration of this method is critical as human amniotic membrane fixation and gelatin embedding steps, are difficult to learn and require a particular handling approach.

engineering-transplantation-suitable-retinal-pigment-epithelium

Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

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

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

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

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

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

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

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

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

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols 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 ...

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 ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch&#039;s Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...