The authors would like to thank the members of Goureau&#39;s team for their input during the set-up of the methods described here, and G. Gagliardi and M. Garita for their critical reading. This work was supported by grants from the ANR (GPiPS: ANR-2010-RFCS005; SightREPAIR: ANR-16-CE17-008-02), the Retina France Association and the technology transfer company SATT Lutech. It was also performed in the frame of the LABEX LIFESENSES (ANR-10-LABX-65) supported by the ANR within the Investissements d&#39;Avenir program (ANR-11-IDEX-0004-02).

The protocol described in this paper follows the guidelines of the Institut de la Vision&#39;s research ethics committee. The Institut de la Vision has been allowed the manipulation of human specimen according to the current French regulation. Specimen handling follows patient data protection in accordance with the Tenets of Helsinki, and national regulations after the ethical approval of the &#34;Comit&#233; de Protection des Personnes (CPP) Ile-de-France V&#34;.
1. Preparation of Culture Media and Dishes
<ol>
	<li>Culture media
	<ol>
		<li>Use iPSC medium, a chemically defined medium dedicated to pluripotent stem cell culture in feeder-free conditions16. Prepare 500 mL of medium according to the manufacturer&#39;s protocol.</li>
		<li>Use Basal iPSC (Bi) medium, the iPSC chemically defined medium without fibroblast growth factor 2 (FGF2) or transforming growth factor Beta (TGF&#223;).</li>
		<li>Prepare 500 mL of Bi medium supplemented with 1% N2 supplement (BiN2 medium), 10 units/mL of penicillin, and 10 mg/mL of streptomycin.</li>
		<li>Prepare 500 mL of proneural N2-based medium (ProN2 medium) composed of DMEM:Nutrient Mixture F-12 (DMEM/F12, 1:1, L-Glutamine), 1% MEM nonessential amino acids, 1% N2 supplement, 10 units/mL of penicillin, and 10 mg/mL of streptomycin.</li>
		<li>Prepare proneural B27-based medium (ProB27 medium) composed of DMEM:Nutrient Mixture F-12 (DMEM/F12, 1:1, L-Glutamine), 1% MEM nonessential amino acids, 2% B27 supplement, 10 units/mL of penicillin and 10 mg/mL of streptomycin.</li>
	</ol>
	</li>
	<li>Culture vessel preparation
	<ol>
		<li>For human iPSC culture
		<ol>
			<li>Prepare 10 mL of a vitronectin solution containing 5 &#956;g/mL of vitronectin in 1x PBS. In 10 mL of 1x PBS, add 100 &#181;L of the thawed vitronectin stock solution (100x).</li>
			<li>Distribute 2 mL of the vitronectin solution per 6-cm culture dish corresponding to 0.5 &#181;g/cm2. Incubate it for 1 h at room temperature (RT). Remove the vitronectin solution by aspiration using a vacuum-aspiration system or a 5 mL pipette.</li>
			<li>Add 4 mL of iPSC medium per 6-cm dish.</li>
			<li>Incubate the dishes in a cell culture incubator at 37&#160;&#176;C and 5% CO2 for a minimum of 30 min before use.</li>
		</ol>
		</li>
		<li>For human iPSC-derived RPE (hiRPE) cell culture</li>
		<li>Use a pluripotent stem cell-qualified matrix or substrate to coat wells according to the manufacturer&#39;s protocol. Add sufficient matrix or substrate to cover the entire growth surface area. For example, put 300 &#181;L of matrix per well in 24-well plate and 3 mL of matrix in a T-25 cm2 flask.</li>
		<li>Incubate the coated culture vessels for a minimum of 1 h at 37&#160;&#176;C. Remove the matrix using vacuum-aspiration systems or a 5 mL pipette.</li>
		<li>Add 1 mL of ProN2 medium per well on 24-well plates and return the culture vessel to the incubator for a minimum of 30 min to warm and equilibrate the medium. For T-25 cm2 flasks, see step 5.2.</li>
	</ol>
	</li>
</ol>
2. Maintenance and Expansion of Human iPSCs
<ol>
	<li>Maintenance of human iPSCs
	<ol>
		<li>Prepare 3 mL of iPSC medium in a 15 mL tube and keep it at RT for a minimum of 15 min before use. Prepare a coated 6-cm dish as described in 1.2.1.</li>
		<li>Thaw a cryogenic sample of human iPSCs from a liquid nitrogen tank or a -150&#160;&#176;C freezer by incubation in a water bath at 37&#160;&#176;C for 30 s.</li>
		<li>Carefully disinfect the cryogenic vial using a disinfectant solution spray. Transfer the thawed human iPSCs from the cryotube to the 15 mL tube containing 3 mL of iPSC medium pre-warmed at RT.</li>
		<li>Centrifuge the tube at 110 x g for 3 min. Remove the supernatant by aspiration using a vacuum-aspiration system or a 5 mL pipette.</li>
		<li>Resuspend the cell pellet with 1 mL of iPSC medium from a vitronectin-coated 6-cm culture dish using a 2 mL pipette and transfer the cells back to the dish. Return the dish to the incubator at 37&#160;&#176;C and 5% CO2 for a minimum of 24 h before changing the medium. 
		NOTE: A ROCK inhibitor, such as Y-27632 at 10 &#181;M, can be added to the iPSC medium in the 6-cm culture dishes to reduce apoptosis.</li>
		<li>Change the medium daily and pass the human iPSCs every week.</li>
	</ol>
	</li>
	<li>Passaging of human iPSCs
	<ol>
		<li>Pass the human iPSC at a 70 - 80% confluence, classically after 7 days in culture.</li>
		<li>Prepare 6-cm dishes for the passaging as described in step 1.2.1. Remove the iPSC medium from the dishes with confluent iPSCs and add 2 mL of a dissociation solution for 6 min at RT. 
		NOTE: The incubation time depends on the iPSC clones. Try a 6 min incubation as a start.</li>
		<li>Remove the dissociation solution using vacuum-aspiration systems or a 5 mL pipette and add 2 mL of iPSC medium pre-warmed at RT. Resuspend the iPSC colonies by pipetting them up and down 5 - 10x with a 1,000 &#181;L pipette. 
		CAUTION: Avoid single cell dissociation from excessive pipetting.</li>
		<li>Transfer 30 to 200 &#181;L of resuspended cell clumps in a new 6-cm culture dish. Return the dishes to the cell culture incubator at 37&#160;&#176;C and 5% CO2 for a minimum of 24 h before changing the medium. 
		NOTE: The volume of resuspended cell clumps needed for the passaging depends on the iPSC clones.</li>
		<li>Change the medium daily and pass the human iPSCs every week.</li>
	</ol>
	</li>
</ol>
3. Generation of Retinal Organoids
<ol>
	<li>Start the iPSC differentiation following the protocol schematized in Figure 1A when the colonies reach a 60 - 70% confluence (Figure 1B).</li>
	<li>Prepare 4 mL of Bi medium per 6-cm dish. Warm the Bi medium to 37&#160;&#176;C. Change the iPSC medium to Bi medium. Note this time as day 0 (D0).</li>
	<li>At D2, switch the cultures in the Bi medium to a BiN2 medium, previously warmed at 37&#160;&#176;C. Change the medium every 2 - 3 days.</li>
	<li>Identify emergent self-forming retinal organoids by neuroepithelium buds, as shown in Figure 1C - 1E. 
	NOTE: These early retinal organoids, corresponding in their developmental stage to the optic cup, can be manually isolated for downstream experimentation.</li>
</ol>
4. Maturation of Retinal Organoids
<ol>
	<li>At D28, prepare 6-well plates containing 4 mL/well of ProB27 medium initially supplemented with 10 ng/mL of FGF2 (Figure 1A). 
	NOTE: Add FGF2 immediately before the media is added to the plates. 
	Caution: Do not filter FGF2.</li>
	<li>Recover the retinal structures from the 6-cm dishes manually. To do this, isolate the structures making perpendicular striations with the needle around the neuroepithelial bud, as shown in Figure 1E, and detach the organoids by gently scratching them with the needle.</li>
	<li>Aspirate 10 - 15 organoids using a 1,000 &#181;L pipette and transfer them in a single well of the 6-well plate containing ProB27 medium.</li>
	<li>Keep the retinal organoids in floating culture conditions in ProB27 medium in a cell culture incubator at 37&#160;&#176;C and 5% CO2. Change half of the medium every 2 - 3 days. 
	NOTE: Treat the organoids with FGF2 until D35.</li>
	<li>At D35, remove half of the medium and add fresh prewarmed ProB27 medium at 37&#160;&#176;C without FGF2.</li>
	<li>Change half of the medium every 2 - 3 days during the time required to obtain the desired retinal cell types according to the emergence of retinal cell types, as depicted in Figure 2.</li>
</ol>
5. Generation and Amplification of Human iPSC-derived RPE (hiRPE) Cells
<ol>
	<li>Generation of human hiRPE cells
	<ol>
		<li>Engage the iPSCs in differentiation when the colonies reach a 60 - 70% confluence, following the protocol schematized in Figure 3A.</li>
		<li>Prepare 4 mL per 6-cm dish of Bi medium and warm the Bi medium at 37&#160;&#176;C. Change the iPSC medium to Bi medium. Note this time as day 0 (D0).</li>
		<li>At D2, switch the cultures in the Bi medium to a BiN2 medium previously warmed at 37&#160;&#176;C. Change the medium every 2 - 3 days.</li>
		<li>At D28, change the medium to a fresh ProN2 medium previously warmed at 37&#160;&#176;C as depicted in Figure 3A. 
		NOTE: This step can be done after the organoid picking described in step 4.</li>
		<li>Change the ProN2 medium every 2 - 3 days.</li>
		<li>At D42, identify emergent hiRPE cells by the observation of pigmented patches as shown in Figure 3B. 
		NOTE: The pigmentation of hiRPE cells appears between D35 to D56 depending on the iPSC clones.</li>
	</ol>
	</li>
	<li>Amplification of hiRPE Cells
	<ol>
		<li>At D42, prepare a 24-well plate previously coated with the matrix as described in step 1.2.2 and containing 1 mL of ProN2 medium per well. Place the plate for 15 min in the incubator at 37&#160;&#176;C and 5% CO2 before use.</li>
		<li>Recover the hiRPE patches from the 6-cm dish manually. To isolate patches, make a perpendicular striation with the needle around the pigmented epithelium and detach the sheet by gently scratching it with the needle. Aspirate 10 pigmented patches using a 1,000 &#181;L pipette and transfer them in a single well of the 24-well plate.</li>
		<li>Keep the hiRPE patches in ProN2 medium in a cell culture incubator at 37&#160;&#176;C and 5% CO2 for 48 h before changing the medium. Note this hiRPE cell passage as 0 (hiRPEp0). Change the medium every 2 - 3 days with ProN2 medium.</li>
		<li>Pass the hiRPEp0 cells when the cells are confluent.
		<ol>
			<li>Remove the medium using vacuum-aspiration systems and wash each well 1x with 1 mL of PBS using a 5 mL pipette.</li>
			<li>Discard the PBS using vacuum-aspiration systems, add 200 &#181;L per well of 0.25% trypsin, and incubate it for a minimum of 15 min in an incubator at 37&#160;&#176;C. Add 800 &#181;L of ProB27 medium to inactivate the trypsin. 
			NOTE: The use of ProN2 medium to stop the trypsin activity is not recommended.</li>
			<li>Dissociate the sheet of hiRPE cells by pipetting it up and down. Place the cell suspension in a 15 mL tube and centrifuge it for 5 min at 110 x g. 
			NOTE: An additional wash can be done with ProN2 medium to remove the excess of ProB27 medium.</li>
			<li>Remove the supernatant and gently resuspend the cell pellet in 2 mL of ProN2 medium pre-warmed at 37&#160;&#176;C. Count the cells with a cell counter.</li>
			<li>Take 1.25 million cells and place them in a matrix-coated T-25 cm2 flask (see step 2.2.2) containing 5 mL of ProN2 medium pre-warmed at 37&#160;&#176;C. Note this hiRPE cell passage as 1 (hiRPEp1).</li>
		</ol>
		</li>
		<li>Keep the hiRPE cells in the ProN2 medium in a cell culture incubator at 37&#160;&#176;C and 5% CO2 for 48 h before changing the medium.</li>
		<li>Change the media every 2 - 3 days. When the hiRPEp1 cells reach confluence, perform the next passage or cryopreservation.</li>
	</ol>
	</li>
</ol>
6. Cryopreservation of Retinal Organoids and hiRPE Cells
<ol>
	<li>For whole retinal organoids
	<ol>
		<li>Select 5 to 20 retinal organoids using a transfer pipet and place them in a cryogenic vial. Remove any excess medium with a 1,000 &#181;L pipette without touching the organoids at the bottom of the tube and add 250 &#181;L of cold cryopreservation medium (see Table of Materials).</li>
		<li>Freeze the vials in an isopropanol-based freezing container at -80&#160;&#176;C for a minimum of 4 h. Transfer the frozen vials to a -150&#160;&#176;C freezer or liquid nitrogen tank for long-term storage.</li>
	</ol>
	</li>
	<li>For hiRPE cells
	<ol>
		<li>At passage 1, dissociate the hiRPE cells when the cells are confluent. 
		NOTE: A cryopreservation of hiRPEp0 cells is not recommended.</li>
		<li>Aspirate the medium from the T-25 cm2 flask using vacuum-aspiration systems and wash it 1x with 3 mL of PBS using a 5 mL pipette.</li>
		<li>Remove the PBS using vacuum-aspiration systems, add 1 mL of 0.25% trypsin, and incubate it for a minimum of 15 min in an incubator at 37&#160;&#176;C. Add 5 mL of ProB27 medium to stop the trypsin activity. 
		NOTE: The use of ProN2 medium to inactivate trypsin is not recommended.</li>
		<li>Dissociate the sheet of hiRPE cells by pipetting it up and down using a 10 mL pipette. Count the cells with cell counter. Place the cell suspension in a 15 mL tube and centrifuge it for 5 min at 110 x g. 
		NOTE: An additional wash can be performed with ProN2 medium to remove the excess of ProB27 medium.</li>
		<li>Aspirate the supernatant using vacuum-aspiration and gently resuspend cells to obtain 2 million cells in 250 &#181;L of cryopreservation medium.</li>
		<li>Transfer 250 &#181;L of cell suspension per cryogenic vial. Freeze the vials in an isopropanol-based freezing container at -80&#160;&#176;C for a minimum of 4 h.</li>
		<li>Transfer the frozen vials to a -150&#160;&#176;C freezer or a liquid nitrogen tank for long-term storage.</li>
	</ol>
	</li>
</ol>
7. Thawing of Retinal Organoids and hiRPE Cells
<ol>
	<li>Thawing of whole retinal organoids
	<ol>
		<li>Warm ProB27 medium at 37&#160;&#176;C (2 mL will be required for each cryogenic vial).</li>
		<li>From the liquid nitrogen tank or -150&#160;&#176;C freezer, thaw a cryogenic vial containing retinal organoids in a water bath at 37&#160;&#176;C for 30 s. Disinfect the cryogenic vial carefully using a disinfectant solution spray.</li>
		<li>Open the vial and add 1 mL of pre-warmed ProB27 medium. Transfer the organoids to a 1.5 mL tube with a transfer pipette.&#160;</li>
		<li>Remove the medium gently by pipetting it without touching the retinal structures at the bottom of the tube. Wash the organoids 1 more time with 1 mL of the pre-warmed ProB27 medium.</li>
		<li>Aspirate the organoids with a transfer pipet and place them in 1 well of a 6-well plate containing ProB27 medium pre-warmed and equilibrated in an incubator at 37&#160;&#176;C and 5% CO2. 
		NOTE: Place 10 - 15 retinal organoids per well of a 6-well plate.</li>
		<li>Change half of the medium every 2 - 3 days during the time required to obtain the desired retinal cell types according to their emergence, as described in Figure 2.</li>
	</ol>
	</li>
	<li>Thawing of hiRPE cells
	<ol>
		<li>Warm ProN2 medium at 37&#160;&#176;C (8 mL will be required for each cryogenic vial).</li>
		<li>From the liquid nitrogen tank or -150&#160;&#176;C freezer, thaw a cryogenic sample of hiRPEp1 cells by an incubation into a water bath at 37&#160;&#176;C for 30 s. Disinfect the cryogenic vial carefully using disinfectant solution spray.</li>
		<li>Open the tube and add 1 mL of pre-warmed ProN2 medium and transfer the cell suspension to a 15 mL tube containing 2 mL of pre-warmed ProN2 medium. Centrifuge it for 5 min at 110 x g.</li>
		<li>Remove the supernatant and gently resuspend the cell pellet in 2 mL of ProN2 medium pre-warmed at 37&#160;&#176;C.</li>
		<li>Count the cells with a cell counter.</li>
		<li>Take 1.25 million cells and place them in the matrix-coated T-25 cm2 flask (see step 2.2.2) containing 5 mL of ProN2 medium pre-warmed at 37&#160;&#176;C. At this stage, note the passage as 2 (hiRPEp2).</li>
		<li>Keep the hiRPEp2 cells in ProN2 medium in the cell culture incubator at 37&#160;&#176;C and 5% CO2 for 48 h before changing the medium.</li>
		<li>Change the media every 2 - 3 days. When the hiRPE cells reach confluence, perform the next passage or cryopreservation. 
		NOTE: After 2 weeks in culture, 8 - 10 million hiRPE cells can be collected for further experimentations.</li>
	</ol>
	</li>
</ol>

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Sacha Reichman, Olivier Goureau, and Jos&#233;-Alain Sahel are inventors on pending patents related to the generation of retinal cells from human pluripotent stem cells.

This protocol describes how to produce RPE cells and retinal organoids, containing retinal RGCs and photoreceptors, from human pluripotent stem cells in xeno-free and feeder-free conditions. Compatible with the Good Manufacturing Practice (GMP) process, the method cultivated presented here allows a large production of iPSC-derived retinal cells as RPE cells, RGCs, and photoreceptors for the development of stem cell-based therapies and drug discovery approaches for the future treatment of retinal degenerative diseases. Th...

The retina is an integral part of the central nervous system (CNS) and has a limited capacity to spontaneously regenerate following a traumatic injury or diseases. Therefore, degenerative pathologies causing definitive retinal cell loss, such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), glaucoma, and diabetic retinopathy, typically lead to irreversible blindness. Rescuing the degenerated retina is a major challenge for which stem cell-based therapies aiming to replace the damaged or lost cells are one of the most promising approaches1,2,3. Pluripotent stem cells as human embryonic stem cells (ESCs) cells or human-induced pluripotent stem cells (iPSCs) have the capacity to be expanded indefinitely in culture, and they have the potential to produce any cell types. Advances in our understanding of retinal development and the improvement of in vitro protocols for human iPSC differentiation have resulted in the generation of retinal organoids7,8,9,10,11,12. All of the major retinal cells, including retinal ganglion cells (RGCs), photoreceptors, and retinal pigmented epithelial (RPE) cells, have been successfully differentiated from human ESCs and iPSCs4,5,6. Based on the SFEB (serum-free culture of embryoid body-like aggregates) method developed by Eiraku et al.13, self-formation of retinal organoids can be obtained from ESC- or iPSC-derived embryoid body-like aggregates in defined extracellular matrix components7,10,14. But these protocols are intricate, requiring a large number of steps not always compatible with the large production of cells for therapeutic approaches or drug screening. Thus, the choice of the method to produce human retinal cells is critical and the method needs to be robust, scalable, and efficient.
Here, based on our previous publication15, we describe each step for a simple and efficient generation of retinal cells through retinal organoid self-formation from adherent human iPSCs cultivated in a feeder-free and xeno-free condition. Starting from routine cultures of adherent human iPSCs, this protocol requires only a simple successive medium changing to allow the generation of both iPS-derived RPE (hiRPE) cells and neuroretinal structures in 4 weeks. After a manual isolation, hiRPE can be expanded and the retinal structures can be cultured as floating organoids where the retinal progenitor cells are able to differentiate into all retinal cell types in a sequential order consistent with the in vivo human retinogenesis. Finally, for research advancement or clinical translation, we describe a cryopreservation method allowing the long-term storage of whole retinal organoids and hiRPE cells without affecting their phenotypic characteristics and functionality.

<table><tbody><tr><td>Vitronectin (VTN-N) Recombinant Human Protein, Truncated</td><td>ThermoFisher Scientific</td><td>A14700</td><td>Coating</td></tr><tr><td>CTS Vitronectin (VTN-N) Recombinant Human Protein, Truncated</td><td>ThermoFisher Scientific</td><td>A27940</td><td>Coating</td></tr><tr><td>Essential 8 Medium</td><td>ThermoFisher Scientific</td><td>A1517001</td><td>medium</td></tr><tr><td>Essential 6 Medium</td><td>ThermoFisher Scientific</td><td>A1516401</td><td>medium</td></tr><tr><td>CTS (Cell Therapy Systems) N-2 Supplement</td><td>ThermoFisher Scientific</td><td>A1370701</td><td>supplement CTS</td></tr><tr><td>N-2 Supplement (100X)</td><td>ThermoFisher Scientific</td><td>17502048</td><td>supplement</td></tr><tr><td>B-27 Supplement (50X), serum free</td><td>ThermoFisher Scientific</td><td>17504044</td><td>supplement</td></tr><tr><td>CTS B-27 Supplement, XenoFree</td><td>ThermoFisher Scientific</td><td>A1486701</td><td>supplement CTS</td></tr><tr><td>DMEM/F-12</td><td>ThermoFisher Scientific</td><td>11320074</td><td>medium</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100X)</td><td>ThermoFisher Scientific</td><td>11140035</td><td>supplement</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>ThermoFisher Scientific</td><td>15140122</td><td>antibiotic</td></tr><tr><td>CellStart CTS</td><td>ThermoFisher Scientific</td><td>A1014201</td><td>Matrix CTS</td></tr><tr><td>Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix</td><td>ThermoFisher Scientific</td><td>A1569601</td><td>Matrix</td></tr><tr><td>Gentle Cell Dissociation Reagent</td><td>Stemcell Technologies</td><td>7174</td><td>dissociation solution</td></tr><tr><td>Cryostem Freezing Media</td><td>clinisciences</td><td>05-710-1D</td><td>Cryopreservation medium</td></tr><tr><td>Fibroblast growth factor 2 (FGF2)</td><td>Preprotech</td><td>100-18B</td><td>FGF2</td></tr><tr><td>Fibroblast growth factor 2 (FGF2) animal free</td><td>Preprotech</td><td>AF-100-18B</td><td>FGF2 Xeno free</td></tr><tr><td>AGANI needle 23G</td><td>Terumo</td><td>AN*2332R1</td><td>Needle</td></tr><tr><td>Flask 25 cm&amp;sup2; Tissue Culture Treated</td><td>Falcon</td><td>353109</td><td>T-25 cm&amp;sup2;</td></tr><tr><td>24 well plate Tissue Culture Treated</td><td>Costar</td><td>3526</td><td>24-well plate</td></tr><tr><td>6 well plate Tissue Culture Treated</td><td>Costar</td><td>3516</td><td>6-well plate</td></tr></tbody></table>

defined xeno-free and feeder-free culture conditions for the generation of human ipsc-derived retinal cell models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The first step for human iPSC differentiation cultivated in feeder-free conditions16 is to shut down self-renewal machinery using Bi medium to encourage a spontaneous differentiation (Figure 1A). Then, at D2, the Bi medium is complemented with an N2 supplement to guide differentiating iPSCs cells towards the neural and retinal lineages. This process leads to the appearance of neuroretinal buds at around D28 (Figure...

Watch this Scientific Journal Video about Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models at JoVE.com

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

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

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

defined-xeno-free-feeder-free-culture-conditions-for-generation-human

Research

JoVE Journal

Developmental Biology

9.2K Views.  Sorbonne Université, INSERM, CNRS, F-75012 Paris, France. 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.

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

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 ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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 ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

Biology

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.

In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority ...

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

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

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

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Enhanced 3D Neural Retina Differentiation from Human Embryonic Stem Cells

Generating Neural Retina from Human Pluripotent Stem Cells

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

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

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

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

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

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

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

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

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