Modeling retinitis pigmentosa through patient-derived retinal organoids

Summary Human-induced pluripotent stem cells (hiPSCs) can be differentiated into well-structured retinal organoids. In this protocol, we successfully established 3D retinae from patient-derived hiPSCs and built the retinitis pigmentosa model in vitro. Moreover, mutation in the retinitis pigmentosa GTPase regulator (RPGR) gene was corrected by CRISPR-Cas9 gene editing, which rescued the structure and function of the 3D retinae. For complete details on the use and execution of this protocol, please refer to Deng et al. (2018).


SUMMARY
Human-induced pluripotent stem cells (hiPSCs) can be differentiated into wellstructured retinal organoids. In this protocol, we successfully established 3D retinae from patient-derived hiPSCs and built the retinitis pigmentosa model in vitro. Moreover, mutation in the retinitis pigmentosa GTPase regulator (RPGR) gene was corrected by CRISPR-Cas9 gene editing, which rescued the structure and function of the 3D retinae. For complete details on the use and execution of this protocol, please refer to Deng et al. (2018).

Reconstitution of reagents
Timing: 2.5-3.0 h 1. 5 mg/mL DNase I (1003) a. Dissolved 15 mg of DNase I in 3 mL of ultra-pure water. b. After mixing well, use a 0.22-mm membrane filter for the sterilizing filtration. c. Aliquot 100 mL of the dissolved DNase I into sterile 200-mL Eppendorf tubes. d. Store the reconstituted solution at À20 C, which will be stable until the expiration date printed on the label. 2. 10 mM Y-27632 (1,0003) a. Centrifuge quickly before opening the lid, 2000 3 g for 30 s, to ensure that all the powder sediments at the bottom of the tube. b. Dissolved 10 mg of Y-27632 in 3.1226 mL of dimethyl sulfoxide (DMSO). c. After complete dissolution, aliquot 20 mL of dissolved Y-27632 into sterile 200-mL Eppendorf tubes. d. Store the reconstituted solution at À80 C where it can remain for up to two years, and the powder can be stored at À20 C for three years. 3. Recombinant human BMP4 (hBMP4) a. Add 10 mg of bovine serum albumin (BSA) powder to 10 mL of ultra-pure water. b. Mix slowly for 30 min, at least, at 25 C until the BSA powder is completely dissolved. c. Filter the solution using a 0.22-mm filter to ensure sterile filtration. d. Centrifuge quickly at 2000 3 g for 30 s, before opening the lid, to ensure that all the powder sediments at the bottom of the tube.
e. Dissolved 50 mg of hBMP4 in sterile 4-mM HCl containing 0.1% BSA. f. After complete dissolution, aliquot 20 mL of dissolved hBMP4 into sterile 200-mL Eppendorf tubes. g. Store the solution at À20 C for up to 6 months. After reconstitution, it can be stored at 2 C to 8 C for up to 1 month, or store at À70 C for up to 3 months. 4. 5 mM retinoic acid (203) a. Dissolve 100 mg of retinoic acid in 3.33 mL of DMSO to obtain a stock solution (100 mM). b. Aliquot and store in light protected vials at À80 C for up to 2 weeks. c. Dilute 100 mM retinoic acid using DMSO to a 5-mM concentration and store the solution in the dark at -20 C for up to 2 weeks. d. Diluted it in tissue culture medium right before use.
CRITICAL: Retinoic acid is more sensitive to light, heat, and air in solution. The prepared retinoic acid stock solution should be stored in the dark until use. (1,0003) a. Add 0.125 g of taurine in to 10 mL of Dulbecco's phosphate buffered saline (DPBS). b. After complete dissolution, aliquot 1 mL of taurine solution into sterile 1.5-mL Eppendorf tubes. c. Store the solution at À20 C until the expiration date printed on the label. 6. Matrigel preparation (protein concentration: 8.1 mg/mL) a. Thaw Matrigel at 4 C for 1-2 days until it melts completely. b. Store the 1.5-mL Eppendorf tubes and pipettes in the À20 C freezer one day in advance to prechill them. c. Aliquot 370 mL of the Matrigel into prechilled 1.5-mL Eppendorf tubes on ice using prechilled pipettes. d. Store the aliquots quickly at À20 C after labeling them.

100 mM Taurine
CRITICAL: Freeze-thaw cycles should be minimized by aliquoting into one-time-use aliquots. Store aliquots at À20 C in the freezer until use.

Reconstitution of the media
Note: Make sure all the media and reagents are prepared under aseptic conditions and warmed at room temperature (20 C-25 C) before use.

Medium reconstitution for urinary cells
Note: Store the primary medium in the dark at 4 C and use it within 2 weeks.
Note: Store the proliferation medium in the dark at 4 C and use it within 2 weeks.
Note: The solution should be freshly reconstituted before use.
Note: The solution should be freshly reconstituted before use. Note: Store the solution at 4 C for up to 1 month.

Medium reconstitution for hiPSCs
Note: Store the medium at 4 C for up to 1 month.
Note: Store the medium at 4 C and use it within 2 weeks.
Note: The solution should be freshly reconstituted before use.

Medium reconstitution for retinal organoid differentiation
Note: The solution should be freshly reconstituted before use. Note: Store the differentiation medium at 4 C for up to 1 month.
Note: Store the medium in the dark at 4 C and use it within 2 weeks.

Establishment of hiPSCs from urine samples
Note: All the previously described procedures (Zhou et al., 2012) for urinary cell isolation and expansion were used with some modifications (Deng et al., 2018).
CRITICAL: Discard the first streams of urine.
CRITICAL: The urine container must be sterile, disposable sterile bottles are recommended.
CRITICAL: It is also recommended that the opened container should not contact volunteers' skin to prevent contamination.
2. Transfer them into 50-mL tubes using a 25-mL pipette as soon as possible.
CRITICAL: Urinary cells should be isolated immediately after urine collection.
3. Centrifuge the samples at 400 3 g for 10 min at room temperature (20 C-25 C). 4. Discard the supernatant carefully until 1 mL or less of the liquid is left in the tube. 5. Resuspend and collect the liquid from one sample into a 50-mL tube. 6. Add 10 mL of washing buffer to dilute the samples. 7. Centrifuge at 200 3 g for 10 min at room temperature (20 C-25 C). 8. During centrifugation, aspirate the gelatin solution of the cell culture plates and wash twice using washing buffer. 9. Add 1 mL of the primary medium into each gelatin-coated well. 10. After centrifugation, discard the supernatant until 0.2 mL or less of the sample is left in the tube. 11. Resuspend the sediment in 1 mL of primary medium. 12. Transfer the cells onto gelatin-coated 6-well plates and then incubate the plates.
CRITICAL: Steps 9-12 should be performed in the dark. CRITICAL: The gelatin-coated plates should be freshly coated; thus, it would better to coat the plates 0.5-1 h before use.
Expansion of urinary cells Day 1, day 2, and day 3: Addition of the primary medium Timing: 0.5 h 13. Add 1 mL of the primary medium to the wells to maintain the concentration of antibiotics and nutrition.

CRITICAL:
Step 13 should be performed in the dark. The following days: Half medium change using the proliferation medium Note: Small urinary cell colonies appeared within 3-5 days after plating and grew steadily.

CRITICAL:
The gelatin-coated plates should be freshly coated; thus, it would better to coat the plates 0.5-1 h before use.
CRITICAL: The cells used for transfection were in a single cell suspension: nucleofection clumps led to a low transfection efficiency.
CRITICAL: A total of 10 5 -10 6 cells from each sample are required for transfection, since a lower or higher number of cells might decrease the transfection efficiency.
27. Then, centrifuge cells at 200 3 g for 3.5 min at room temperature (20 C-25 C). 28. Aspirate the supernatant as much as possible by using pipette tips. 29. Resuspend cells in 100 mL of nucleofection solution. 30. Gently transfer the sample to Nucleocuvette vessels and tap the vessels to make sure the sample is at the bottom of the vessels.
CRITICAL: Tap the Nucleocuvette vessels gently after the mixture is added, to avoid the presence of air bubbles and to completely cover the bottom of the cuvette with the sample.

OPEN ACCESS
STAR Protocols 2, 100438, June 18, 2021 31. Transfer the vessels to the retainer and electroporate them using the 4D-nucleofector system by using the T-020 program. Troubleshooting 1 32. Finally, after the electroporation procedure, gently transferred the urinary cells into Matrigelcoated plates containing TeSR-E8 medium.
CRITICAL: The Matrigel-coated plates should be freshly coated; thus, it would better to coat the plates 0.5-1 h before use.
33. Change the medium after 24 h of transfection. 34. Grow the transfected urinary cells in a hypoxia chamber (5%-6% O 2 ) until the human ESC-like colonies appear ( Figure 1C).

hiPSC colony isolation and expansion
Timing: 1 h 35. Approximately three weeks post-electroporation, isolate hiPSC colonies according to following steps. First, break the hiPSC colonies into a checkerboard-shaped grid by using sharp needles under an inverted microscope ( Figure 1D). Troubleshooting 2 CRITICAL: Pick only well-separated and hESC-like colonies to make sure they are clones. Representative pictures of "good" colonies and "bad" colonies are shown in Figure 2.
36. Then, collect the colony pieces using 100-mL pipette tips and transfer them to new Matrigelcoated plates containing fresh TeSR-E8 medium supplemented with 10 mM Y-27632.
CRITICAL: One colony should be seeded per well. Each time, when a colony is picked, the needle and tip should be replaced.
CRITICAL: The Matrigel-coated plates should be freshly coated; thus, it would better to coat the plates 0.5-1 h before use.

OPEN ACCESS
37. RPGR mutation is used for hiPSC correction (Figure 3). Use the CRISPR sgRNA design tool to design sgRNA target exon 14 near the mutation site. 38. Dissolve both oligos to 100 mM using nuclease-free water. 39. Phosphorylate and anneal the two oligos using the following conditions: 37 C for 30 min, 95 C for 5 min and then bring the temperature down to 25 C at 5 C/min.
CRITICAL: Set the thermal cycler's heated lid to 75 C to prevent samples from evaporating and drying out.
40. In the meantime, digest the pX330 plasmid using BbSI at 37 C for 30 min.
Alternatives: Water bath can be used instead of the thermal cycler for heating.
41. Purify the digested pX330 plasmid using a 1% agarose gel and QIAquick Gel Extraction Kit, following the manufacturer's instructions.
CRITICAL: 1% agarose gel should be freshly reconstituted before use.
42. Detect the concentration of the digested pX330 plasmid using NanoDrop device. 43. Then, set up a ligation reaction and incubate the digested pX330 plasmid and sgRNA at room temperature (20 C-25 C) for 10 min. 44. Transform the product of ligation into competent cells following the manufacturer's instructions. CRITICAL: The product of the ligation reaction should be stored at À20 C or on ice before use.
CRITICAL: After transformation, pick only well separated and round colonies to make sure they are clones.
CRITICAL: Keep the Luria-Bertani (LB) agar plate containing colonies at 4 C for no more than one month after sealing, in order be able to isolate more colonies, if necessary.
45. After amplification, extract the plasmid DNA containing the target sequence using QIAGEN En-doFree Plasmid Maxi Kit, by following the manufacturer's instructions. 46. Store it at À20 C before use. 47. Analyze the plasmid DNA using Sanger sequencing.
Pause point: The plasmid DNA may be safely stored in the freezer until convenient.
CRITICAL: The plasmid DNA can be store at À20 C for up to 6 months, but freeze-thaw cycles should be avoided.

Donor DNA design and plasmid cloning
Timing: 2 weeks 48. As a homologous recombinant template, amplify donor exon 14 from the normal template. Homology arms and a selection cassette are recommended. 49. After amplified and purified, insert a 3.4 kbp homology directed repair (HDR) fragment carrying a selection cassette of neomycin (G418) into pEASY-Blunt simple cloning vector following pEASY-Blunt cloning protocol.
CRITICAL: After transformation, pick only well separated and round colonies to make sure they are clones.
Pause point: Maintain the LB agar plate containing colonies at 4 C for no more than one month after sealing, in order to be able to isolate more colonies, if necessary.
50. Analyze the colonies using Sanger sequencing. 51. After amplification, extract the plasmid DNA by using EndoFree Plasmid Mini Kit, following the manufacturer's instructions. 52. Store the plasmid DNA at À20 C before use.
Pause point: The plasmid DNA may be safely stored in the freezer until convenient.

Plasmid electroporation
Timing: 1 hour 53. When the hiPSCs show 70%-80% confluence, use accutase to dissociate hiPSCs into single cells. 54. Harvest and count the cells, centrifuge the required number of cells, and completely remove the supernatant. 55. For the genome correction knock-in, mix 2 mg of the constructed pX330 plasmid and 2 mg of the targeting vector in nucleofector solution before electroporation. 56. Resuspend the required number of cells in the nucleofector solution.

OPEN ACCESS
CRITICAL: A total of 2-3 3 10 5 cells from each sample are required, since a lower or higher number of cells may decrease the transfection efficiency.
CRITICAL: Tap the Nucleocuvette vessels gently after the mixture is added, to avoid the presence of air bubbles and to completely cover the bottom of the cuvette with the sample.
Note: It has been reported that using Y-27632 may increase the viability of stem cells (Watanabe et al., 2007).
CRITICAL: The vessels should be carefully removed from the retainer after electroporation.
CRITICAL: The Matrigel-coated plates should be freshly coated; thus, it would better to coat the plates 0.5-1 h before use.

Neomycin selection
Timing: 2 weeks 60. When the transfected cells reach 70%-80% confluence, add G418 to TeSR-E8 medium at a final concentration of 200 mg/mL for cell selection. 61. Wash the cells with DPBS and refresh the medium daily.

Genome correction clone screening using PCR and sequencing
Timing: 2 weeks Collect the DNA 62. After selection, G418-insensitive hiPSC colonies were isolated and expanded. 63. Remove the TeSR-E8 medium, rinse the hiPSCs twice with DPBS, and dissociate the cells using an EDTA solution for 5 min. 64. Remove the EDTA and resuspend hiPSCs in fresh TeSR-E8 medium. 65. Centrifuge the suspension at 200 3 g for 5 min and remove the medium.
CRITICAL: Set the thermal cycler's heated lid to 75 C to prevent samples from evaporating and drying out.
68. Finally, perform an extension for 10 min at 72 C and hold the product at 4 C. 69. Verify the PCR product using Sanger Sequencing.
Pause point: The PCR product may be safely stored in the freezer until convenient.
CRITICAL: The final product of PCR can be stored at À20 C for up to 6 months, but freezethaw cycles should be avoided.

Maintenance of hiPSC colonies hiPSC recovery
Timing: 0.5 h 70. Remove the cell lines from liquid nitrogen and thaw them in a 37 C water bath as soon as possible. Troubleshooting 3 CRITICAL: Put the cell lines in water bath immediately after taking it out of the liquid nitrogen.

CRITICAL:
The Matrigel-coated plates should be freshly coated; thus, it would better to coat the plates 0.5-1 h before use.
Alternatives: For hiPSCs maintenance, ncEpic hPSC Medium can be used instead of TeSR-E8 medium.

Medium change
Timing: 0.5 h 75. Aspirate the medium in the 6-well plates and wash the hiPSCs with DPBS once or twice. 76. Add 2-3 mL of TeSR-E8 medium to the plate and incubate them.

Passaging and cryopreservation of hiPSCs
Timing: 0.5 h 77. When cell confluence reaches 70% or more, passage the cells. Aspirate the medium in the 6-well plates and wash the hiPSCs with DPBS once. 78. Add 1 mL of EDTA to digest the hiPSCs at 37 C for 5 min. 79. For passaging, aspirate the EDTA and resuspend hiPSCs in TeSR-E8 Medium containing 10 mM Y-27632.
Note: It has been reported that using Y-27632 may increase the viability of stem cells (Watanabe et al., 2007).
80. Break hiPSC clumps into smaller pieces using gentle pipetting, and add an appropriate volume of cells into a new Matrigel-coated 6-well plate.
CRITICAL: The hiPSC used for passaging should not in a single cell suspension, single cell led to a poor pluripotency status.
81. Passage the cells every third or fourth day. 82. For cell freezing, aspirate the EDTA (from step 78) and resuspend hiPSCs with cell cryopreservation solution. 83. Transfer hiPSCs in a freezing tube and perform gradient cooling. 84. Store the hiPSCs in liquid nitrogen until use.
Pause point: The hiPSCs can be stored in liquid nitrogen until use.

Generation of retinal organoids from hiPSCs
Note: All the previously described procedures (Kuwahara et al., 2015) for retinal organoid differentiation were used with slight modifications Liu et al., 2020). 97. Change half of the differentiation medium to achieve a lower concentration of hBMP4 (0.375 nM; final concentration: 13.75 ng/mL). 98. Aspirated 60 mL of the medium from each well and add 60 mL of the differentiation medium supplemented with 20 mM Y-27632. 99. Incubate the plates in CO 2 incubator.
Day 18: Transfer to non-stick petri dishes Timing: 1-2 h 100. Transfer the aggregates into a Petri dish. 101. Cut them into 2 to 3 pieces using a V-Lance Knife. 102. Aspirate all the aggregates into 15-mL centrifuges tubes. 103. Remove the supernatant after all the aggregates settle at the bottom of the tubes. Resuspend the aggregates in neural retina medium and transfer them into 9-cm non-stick Petri dishes.
CRITICAL: Retinoic acid is more sensitive to light: minimize the light exposure to prevent its isomerization. CRITICAL: Step 103 should be performed in the dark.

Long-term culture of neural retinae
Timing: 0.5 h 104. Refresh the medium every 5 days and protect the culture from light. Troubleshooting 5 CRITICAL: Retinoic acid is more sensitive to light: minimize the light exposure to prevent its isomerization.

CRITICAL:
Step 143 should be performed in the dark.

EXPECTED OUTCOMES
We successfully recapitulated RP predisposed by the RPGR mutation, using patient-derived retinae in a dish. The defects in patient hiPSC-derived retinae are consistent with those in their clinical phenotype. Significant defects of photoreceptor and shorted cilium were found in patient retinal organoids. The photoreceptor structure and ciliopathy were rescued by CRISPR-Cas9-mediated correction of RPGR mutation (Figure 4). This protocol studied RP in vitro utilizing RP patient-derived 3D retinal organoids.

LIMITATIONS
Our protocol includes urinary cell reprogramming, CRISPR-Cas9-mediated genome editing, and retinal organoids differentiation. We not only successfully established hiPSCs from RP patients with the RPGR mutation but also achieved mutation correction using the CRISPR-Cas9 technology. The steps of the differentiation of human retinal organoids are based on the previously published protocol (Kuwahara et al., 2015), with slight modifications. Furthermore, retinal organoids derived from patient-specific hiPSCs rebuild the occurrence and development of RP in vitro. Thus, they are an ideal model for future disease treatment and drug discovery (Jin et al., 2019).