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  • Retinal organoid differentiation from iPSCs cultured in the Cellartis DEF-CS 500 Culture System
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Tech Note

Retinal organoid differentiation from iPSCs cultured in the Cellartis DEF-CS 500 Culture System

Data kindly provided by Dr. Magdalena Renner and Dr. Yanyan Hou, Institute of Molecular and Clinical Ophthalmology Basel, Switzerland.

In this technical note, we define a protocol for retinal organoid differentiation from iPSCs using the Cellartis® DEF-CS™ 500 Culture System. Successful differentiation was supported by the characterization of acquired highly organized human retinal organoids. These retinal organoids can potentially be implemented for drug discovery and studies understanding basic biology.

Introduction Methods Results Conclusion References

Introduction  

The advent of organoids in disease modeling and drug discovery

Some developmental and disease processes are difficult to study in animal models, patients, immortalized cell lines, or cultures of human tissue explants. The development of human organoids—complex, 3D systems generated in vitro from human stem cells—enables the study of human-specific biological processes. Organoids derived from induced pluripotent stem cells (iPSCs) opened new avenues for organ-level disease modeling. With the capability of mass production, organoid techniques are also useful for screening libraries of chemical compounds for drug discovery. However, there is a need to optimize cell culture conditions to optimally drive stem cell proliferation and differentiation into organoids.

In one proof-of-concept study, organoids derived from patients with retinitis pigmentosa (RP) were genetically corrected using CRISPR-Cas9.1 Healthy organoids generated this way might be transplanted back into patients for personalized regenerative medicine.

Challenges and recent advances in retinal modeling

The retina is composed of several layers that transduce light into neuronal signals via a series of biochemical reactions. Methodological advances have made it possible to induce human iPSC to recreate key retina structures and functional features in vitro.2 These simplified versions of retinas can empower developmental research, disease modeling, and testing therapies for retinal diseases such as age-related macular degeneration (AMD) and RP.

Limitations of organoid technology include the requirement for refining culture medium for long-term expansion.3 Retinal organoid reproducibility can also be a limitation; differentiation protocols must also be further optimized along with tools to specifically investigate retinal organoid quality for applications.4 One way of evaluating the quality of retinal organoids is using antibodies against markers of particular cell types. Examples include anti-arrestin 3 for cones, anti-rhodopsin for rods, anti-NRL for nucleus of rods, anti-TRPM1 for rod bipolar cells—post-synapse, anti-OneCut2 for horizontal cell nuclei, and anti-Sox9 for Müller glia.

In this work, retinal organoids were differentiated from iPSCs cultured in Cellartis® DEF-CS™ 500 Culture System following a previously published protocol,5 then evaluated using staining and imaging.

Methods  

Cell culture and generation of retinal organoids from human iPSCs

Retinal organoids were generated from human iPSCs as described before2 with some modifications—the AMASS (agarose microwell array seeding and scraping) protocol5 (Figure 1). Briefly, agarose microwell arrays were prepared in MicroTissues 3D Petri Dish micro-mold spheroids (Sigma) with 500 μl of 2% agarose (Thermo Fisher Scientific) in DMEM with GlutaMax (GIBCO). The solidified molds were transferred to 12-well plates (Corning) and equilibrated with 1.5 mL Cellartis DEF-CS 500 Basal Medium with Additives (Cat. # Y30017).

iPSCs were seeded into the agarose microwell arrays with 300 or 600 cells per microwell in 150 μl DEF-CS 500 medium. The plates were placed in the incubator for 30 minutes to allow the cells to settle into the microwells 1.5 mL DEF-CS 500 medium was gently added to completely cover the agarose mold. The forming embryoid bodies were incubated at 37°C, 5% CO2. Over three days, the medium was replaced with NIM (neural induction medium). On day 7, following culture in NIM, embryoid bodies from one 9x9 mold were transferred to a 6-well plate coated with Matrigel (Corning). On day 16, NIM was exchanged for '3:1 medium’ containing 3 parts DMEM (GIBCO) per 1 part F12 medium (GIBCO), supplemented with 1% B27 without vitamin A (GIBCO), 1% NEAA Solution, 1% penicillin/streptomycin (GIBCO).

On day 28–30 retinal structures were detached from the Matrigel plate by checkerboard scraping. From day 42, aggregates were cultured in 3:1 medium supplemented with an additional 10% heat-inactivated FBS (Millipore) and 100 μM taurine (Sigma) with media changes every other day. At week 10, the culture medium was supplemented with 1 μM retinoic acid (Sigma). From week 14, the B27 supplement in 3:1 media was replaced by N2 supplement (GIBCO) and retinoic acid was reduced to 0.5 μM. Subsequently, histology was performed to characterize retinal organoids.

Figure 1. Differentiation protocol.

Staining and imaging of sectioned organoids

Staining of organoids was begun with a 1-hour incubation in blocking buffer A at room temperature. Buffer A was made by adding the following elements to PBS: 10% normal donkey serum (Sigma), 1% (wt / vol) bovine serum albumin (BSA; Sigma), 0.5% Triton X-100 (Sigma), and 0.02% sodium azide (Sigma). Next, organoid sections were incubated in a humidified chamber with 200 μl solution of primary antibody solution diluted in blocking buffer B (PBS supplemented with 3% normal donkey serum, 1% BSA, 0.5% Triton X-100 and 0.02% sodium azide) at the following dilutions overnight at 4°C: anti-arrestin 3 (1:500), anti-TRPM1 (1:200), anti-Sox9 (1:500), anti-rhodopsin (1:500), anti-NRL (1:200), anti-OneCut2 (1:100)and anti-parvalbumin (1:400). After washing 3 × in PBS supplemented with 0.05% Triton X-100 at room temperature, organoids were incubated with secondary antibodies (Thermo Fisher Scientific, anti-donkey antibodies conjugated to Alexa Fluor 488, 568, or 647) diluted 1:500 in blocking buffer B for 2 hours at room temperature and washed 3 × in PBS-T. Organoids were imaged on an inverted spinning disk confocal microscope (Olympus + Yokogawa CSU W1 Dual camera T2 spinning disk confocal scanning unit).

Results  

Development of retinal organoids started with the generation of embryoid bodies from iPSCs cultured in the DEF-CS culture system. To generate homogenously sized embryoid bodies, we used agarose microarray well technologies that significantly reduce size variability (Figure 1). We could efficiently generate organoids with retinal structures by week 6 of differentiation (Figure 2). The differentiation of the retinal organoids continued until week 30. The organoids were fixed, stained with respective markers, and imaged using spinning disk confocal microscopy. At week 30, the organoids expressed retinal cell type markers such as arrestin 3 (cones), rhodopsin (rods), NRL (rods), and TRPM1 (rod bipolar cells), parvalbumin (horizontal cells), OneCut 2 (horizontal cells), and Sox9 (Müller glia) (Figure 3 and 4). The organoids also had clear outer segments as indicated by the rhodopsin staining.

Figure 2. iPSCs cultured in DEF-CS generated distinct retinal organoids. Phase contrast microscopy images, captured 6 weeks after initiation of differentiation, show a high incidence of organoids containing the retinal component (black spots indicated by arrows are retinal pigment epithelium, RPE).

 

Figure 3. Retinal organoids differentiated from iPSC cultured in DEF-CS develop cones, bipolar cells, and Müller glia. Confocal microscopy images of retinal organoids, captured 30 weeks after initiation of differentiation, show expression of cone arrestin 3, TRPM1, and Sox9, markers specific for cones, rod bipolar cells, and Müller glia, respectively.

 

Figure 4. Retinal organoids differentiated from iPSC cultured in DEF-CS express markers for rods and horizontal cells. Confocal microscopy images of retinal organoids, captured 30 weeks after initiation of differentiation, show expression of Rhodopsin and NRL (markers of rods) along with Parvalbumin and OneCut2 (markers of horizontal cells).

Conclusion  

DEF-CS cultured iPSCs formed retinal organoids, as indicated by the presence of retinal components at 6 weeks of differentiation, observed by phase contrast microscopy. The high quality of these organoids was demonstrated by the expression of markers for cones, bipolar cells, rods, horizontal cells, and Müller glia. The characteristics observed in these retinal organoids indicate iPSCs cultured in Cellartis® DEF-CS™ 500 Culture System are high-quality starting material to get organoids as close to the endogenous organ state as possible.

DEF-CS-cultured iPSCs contribute significantly to the development of diverse human-like in vitro culture systems that could replace the usage of animal systems for drug discovery and studies understanding basic biology.

References  

  1. Deng, Wen-Li et al. “Gene Correction Reverses Ciliopathy and Photoreceptor Loss in iPSC-Derived Retinal Organoids from Retinitis Pigmentosa Patients.” Stem cell reports vol. 10,4 (2018): 1267-1281. doi:10.1016/j.stemcr.2018.02.003
  2. Zhong, Xiufeng et al. “Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.” Nature Communications vol. 5 4047. 10 Jun. 2014, doi:10.1038/ncomms5047
  3. Xu, Hanxiao et al. “Organoid technology in disease modelling, drug development, personalized treatment and regeneration medicine.” Experimental hematology & oncology vol. 7 30. 5 Dec. 2018, doi:10.1186/s40164-018-0122-9
  4. Xue, Y., Lin, B., Chen, J., Tang, W., Browne, A., & Seiler, M. (2022). The Prospects for Retinal Organoids in Treatment of Retinal Diseases. Asia-Pacific Journal of Ophthalmology (Philadelphia, Pa.), 11(4), 314-327. http://dx.doi.org/10.1097/apo.0000000000000538
  5. Cowan, Cameron S et al. “Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.” Cell vol. 182,6 (2020): 1623-1640.e34. doi:10.1016/j.cell.2020.08.013

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C001 This product is manufactured and sold by Takara Bio Europe SAS based on a commercial license to certain intellectual property rights held by Wisconsin Alumni Research Foundation (“WARF”). This product is covered by one or more claims of U.S. Patent No. 7,514,260 and its foreign counterparts. The purchase of this product conveys to the buyer the non-transferable right to use the product for its intended use, strictly limited to purchaser’s own internal research. No other express or implied license is granted to the purchaser. Purchaser cannot have any right to use this product or its components in humans for any purposes including but not limited to diagnostics and/or therapeutics, or otherwise clinical trials. Purchase does not include any right to resell or transfer this product to a third party regardless of whether or not compensation is received. Purchasers wishing to use this product for purposes other than internal research use should contact us.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Y30017: Cellartis DEF-CS 500 Basal Medium with Additives

Y30017: Cellartis DEF-CS 500 Basal Medium with Additives

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