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&#178; Tissue Culture Treated</td>
			<td>Falcon</td>
			<td>353109</td>
			<td>T-25 cm&#178;</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

Universidad de Cartagena UDEC

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

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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 ...