Name: defined xeno-free and feeder-free culture conditions for the generation of human ipsc-derived retinal cell models
Uploaded: 2018-09-06T13:00:00
Description: 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

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...

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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 ...

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