Protocol for purifying iPSC-derived retinal pigment epithelial cells by sequential digestion and intrinsic autofluorescence sorting

Summary

Induced pluripotent stem cells (iPSC)-derived retinal pigment epithelium cells serve as a promising cell source for transplantation therapy in age-related macular degeneration. Here, we present a protocol for purifying the mature retinal pigment epithelium (RPE) cells post-differentiation. We describe steps for sequentially digesting cells with different enzymes and sorting them based on the autofluorescence of mature RPE cells, avoiding fixation and immunostaining. By maintaining high cell viability and purity, this cost-efficient protocol has broad implications for both in vitro research and transplantation applications.

Graphical abstract

Highlights

• •Differentiation of iPSCs into RPE cells guided by growth factors and small molecules
• •Sequential digestion of iPSC-induced RPE cells with papain and Accutase
• •Flow cytometric sorting based on mature RPE autofluorescence
• •Isolation of viable, pure iPSC-RPE without immunostaining, reducing costs

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Induced pluripotent stem cells (iPSC)-derived retinal pigment epithelium cells serve as a promising cell source for transplantation therapy in age-related macular degeneration. Here, we present a protocol for purifying the mature retinal pigment epithelium (RPE) cells post-differentiation. We describe steps for sequentially digesting cells with different enzymes and sorting them based on the autofluorescence of mature RPE cells, avoiding fixation and immunostaining. By maintaining high cell viability and purity, this cost-efficient protocol has broad implications for both in vitro research and transplantation applications.

Before you begin

Age-related macular degeneration (AMD) is the leading cause of vision impairment among the elderly. At present, transplantation therapy shows great promise as a treatment for AMD.¹ Several clinical trials have shown that subretinal transplantation of retinal pigment epithelium (RPE) cells derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) can help maintain or restore vision in patients.^2,3,4 These findings establish a foundation for advancing cell-replacement therapy for AMD. While existing protocols for the differentiation, assessment, and purification of RPE cells are established,^5,6,7,8 further optimization is necessary to enhance safety and efficiency while reducing costs. A cost-effective, high-efficiency protocol will benefit both therapeutic applications and related research endeavors.

In this protocol, we describe an efficient and cost-effective approach for the differentiation and purification of RPE cells. We begin with the culture of iPSCs, detailing procedures for thawing, passaging, and freezing. Next, we describe the differentiation of iPSCs into RPE cells using small molecule reagents and recombinant growth factors. We then outline the assessment of RPE cells maturity by immunofluorescence. Following this, we detail a sequential digestion process using Papain and Accutase to digest intercellular connections. Finally, we describe the flow cytometry-based sorting of cells according to the spontaneous fluorescence of mature RPE cells, eliminating the need for antibody staining and thereby reducing processing time and costs. Adhering to this protocol yields purified mature RPE cells with high viability and purity for further research.

Innovation

After differentiation from iPSCs, mature RPE cells typically establish a basement membrane layer between the cells and the culture plate, as well as tight junctions between adjacent cells. These connections complicate enzymatic digestion when adhering to traditional protocols. To address this challenge, we have developed a sequential digestion protocol utilizing two enzymes: Papain and Accutase. Papain targets the basement membrane, while Accutase specifically addresses cell-cell junctions. This innovative approach significantly enhances both the efficiency of digestion and the viability of cells post-digestion.

Previous protocols for achieving the purity of mature RPE cells generally involve fixation and antibody staining prior to FACS, which are always time-consuming and incur additional costs for antibodies. Moreover, fixed cells are unsuitable for subsequent in vivo applications. Consequently, many earlier methods rely on manual selection of target cells using a needle under microscopic observation when preparing cells for transplantation, raising concerns about the reliability of purity. In contrast, our protocol employs flow cytometry to sort cells based on the autofluorescence of mature RPE cells. This method allows for immediate sorting after digestion without staining, thereby minimizing processing time and eliminating antibody costs. Additionally, this approach enhances cell purity while maintaining high viability.

Institutional permissions

The human iPSC line was obtained from Chengdu Yunce Medical Biotechnology Co., Ltd. The original derivation of this line was performed with full informed consent from the donor. Our specific use of this iPSC line for the current research project was reviewed and approved by the Institutional Review Board of Sichuan Provincial People’s Hospital. All procedures adhered to the guidelines of the International Society for Stem Cell Research (ISSCR) and relevant national regulations for human stem cell research. This iPSC line is approved for research use only and is not intended for clinical application.

Prepare reagents for iPSC culture

Timing: 1 h

• 1.
  Prepare 10 mM Y-27632 stock solution.

  • a.
    Centrifuge the tube containing Y-27632 dihydrochloride powder at 500 × g for 5 min.
  • b.
    Add 1.561 mL of culture-grade H₂O to 5 mg of Y-27632 dihydrochloride powder.
  • c.
    Vortex the centrifuge tube to completely dissolve the powder.
  • d.
    Aliquot 100 μL of the stock solution per tube into 1.5 mL sterile centrifuge tubes.
  • e.
    Store the aliquots at −80°C up to one year.
• 2.
  Prepare mTeSR1 complete medium.

  • a.
    Thaw the mTeSR1 5× supplement for 12–16 hours at 4°C.
  • b.
    Mix the supplement by inverting the bottle, and aliquot 10 mL of the solution per tube into 15 mL sterile centrifuge tubes.
  • c.
    Store the aliquots at −20°C up to six months.
  • d.
    Thaw one aliquot of the 10 mL 5× supplement and add it to 40 mL of mTeSR1 basal medium.
  • e.
    Mix the complete medium by pipetting up and down with a 10 mL pipette.
  • f.
    Store the complete medium at 4°C and use within one week.

Prepare reagents for RPE differentiation

Timing: 1–2 days

• 3.
  Prepare 1 M Nicotinamide (NIC) stock solution.

  • a.
    Weigh 1.221 g of Nicotinamide powder and transfer it to a 15 mL sterile centrifuge tube.
  • b.
    Add 8 mL of culture-grade water and vortex to completely dissolve the Nicotinamide.
  • c.
    Adjust the final volume to 10 mL with culture-grade water and mix again by vortexing.
  • d.
    Sterilize the solution by filtering through a 0.22 μm filter into a new 15 mL sterile centrifuge tube.
  • e.
    Aliquot 1 mL of the NIC solution into 1.5 mL sterile centrifuge tubes.
  • f.
    Store the aliquots at −80°C for up to six months.
• 4.
  Prepare 0.1% BSA in DPBS.

  • a.
    Weigh 50 mg of BSA powder and transfer it to a 50 mL sterile centrifuge tube.
  • b.
    Add 50 mL of DPBS to the tube and vortex until the BSA is completely dissolved.
  • c.
    Filter the 0.1% BSA solution through a 0.22 μm filter into a new 50 mL sterile centrifuge tube.
  • d.
    Store the 0.1% BSA solution at 4°C.
• 5.
  Prepare 100 μg/mL Activin-A stock solution.

  • a.
    Centrifuge the tube containing Activin-A powder at 500 × g for 5 min.
  • b.
    Add 500 μL of 0.1% BSA to 50 μg of Activin-A powder.
  • c.
    Mix the Activin-A stock solution by slowly pipetting up and down with a 1 mL pipette.
  • d.
    Aliquot 100 μL of the stock solution per tube into 1.5 mL sterile centrifuge tubes.
  • e.
    Store the aliquots at −80°C for up to six months.
• 6.
  Prepare 100 μg/mL IGF-1 stock solution.

  • a.
    Centrifuge the tube containing IGF-1 powder at 500 × g for 5 min.
  • b.
    Add 2 mL of 0.1% BSA to 200 μg of IGF-1 powder.
  • c.
    Mix the IGF-1 stock solution by slowly pipetting up and down with a 1 mL pipette.
  • d.
    Aliquot 100 μL of the stock solution per tube into 1.5 mL sterile centrifuge tubes.
  • e.
    Store the aliquots at −80°C for up to six months.
• 7.
  Prepare 100 μg/mL DKK-1 stock solution.

  • a.
    Centrifuge the tube containing DKK -1 powder at 500 × g for 5 min.
  • b.
    Add 1 mL of 0.1% BSA to 100 μg of DKK -1 powder.
  • c.
    Mix the DKK -1 stock solution by slowly pipetting up and down with a 1 mL pipette.
  • d.
    Aliquot 100 μL of the stock solution per tube into 1.5 mL sterile centrifuge tubes.
  • e.
    Store the aliquots at −80°C for up to six months.
• 8.
  Prepare 100 μg/mL Noggin stock solution.

  • a.
    Centrifuge the tube containing Noggin powder at 500 × g for 5 min.
  • b.
    Add 250 μL of 0.1% BSA to 25 μg of Noggin powder.
  • c.
    Mix the Noggin stock solution by slowly pipetting up and down with a 1 mL pipette.
  • d.
    Aliquot 50 μL of the stock solution per tube into 1.5 mL sterile centrifuge tubes.
  • e.
    Store the aliquots at −80°C for up to six months.
• 9.
  Prepare 50 μg/mL bFGF stock solution.

  • a.
    Centrifuge the tube containing bFGF powder at 500 × g for 5 min.
  • b.
    Add 1 mL of 0.1% BSA to 50 μg of bFGF powder.
  • c.
    Mix the bFGF stock solution by slowly pipetting up and down with a 1 mL pipette.
  • d.
    Aliquot 100 μL of the stock solution per tube into 1.5 mL sterile centrifuge tubes.
  • e.
    Store the aliquots at −80°C for up to six months.
• 10.
  Prepare 10 mM SU5402 stock solution.

  • a.
    Add 1.687 mL of DMSO to 5 mg of SU5402 powder.
  • b.
    Vortex the SU5402 stock solution for 3 min to completely dissolve the powder.
  • c.
    Aliquot 100 μL of the stock solution per tube into 1.5 mL sterile centrifuge tubes.
  • d.
    Store the aliquots at −80°C for up to six months.
• 11.
  Prepare 3 mM CHIR99021 stock solution.

  • a.
    Add 1 mL of DMSO to 5 mg of CHIR99021 powder, and transfer into a 5 mL sterile centrifuge tube.
  • b.
    Add another 2.582 mL of DMSO to bring the total volume to 3.582 mL.
  • c.
    Vortex the CHIR99021 stock solution several times. If the powder does not dissolve completely, sonication may be applied.
  • d.
    Aliquot 200 μL of the stock solution per tube into 1.5 mL sterile centrifuge tubes.
  • e.
    Store the aliquots at −80°C for up to six months.

Note: In step b, use the fresh DMSO to rinse the storage tube.

• 12.
  Prepare 50 mg/mL Taurine stock solution.

  • a.
    Weigh 0.5 g of Taurine powder and transfer it to a 15 mL sterile centrifuge tube.
  • b.
    Add 8 mL of culture-grade water to the tube, and vortex to completely dissolve the Taurine.
  • c.
    Add culture-grade water to adjust the final volume to 10 mL, and vortex again to completely mix.
  • d.
    Filter the Taurine solution through a 0.22 μm filter into a new 15 mL sterile centrifuge tube.
  • e.
    Aliquot 1 mL of the Taurine solution per tube into 1.5 mL sterile centrifuge tubes.
  • f.
    Store the aliquots at −80°C for up to six months.
• 13.
  Prepare 20 μg/mL Hydrocortisone stock solution.

  • a.
    Centrifuge the tube containing Hydrocortisone powder at 500 × g for 5 min.
  • b.
    Add 1 mL of DMSO to 50 mg of Hydrocortisone powder and transfer into a 15 mL sterile centrifuge tube.
  • c.
    Add another 4 mL of DMSO to reach a total volume of 5 mL.
  • d.
    Vortex the Hydrocortisone stock solution several times. If the powder does not dissolve completely, sonication may be applied.
  • e.
    Aliquot 1 mL of the 10 mg/mL Hydrocortisone stock solution per tube into 1.5 mL sterile centrifuge tubes.
  • f.
    To prepare the 20 μg/mL stock, dilute 20 μL of the 10 mg/mL Hydrocortisone stock solution into 9.98 mL of DMSO and mix thoroughly by vortexing.
  • g.
    Aliquot 1 mL of the 20 μg/mL Hydrocortisone stock solution per tube into 1.5 mL sterile centrifuge tubes.
  • h.
    Store the aliquots at −80°C for up to six months.
• 14.
  Prepare 10 ng/mL Triiodothyronine stock solution.

  • a.
    Prepare 0.1M NaOH solution.

    • i.
      Weigh 200 mg of NaOH flakes and transfer them to a 50 mL sterile centrifuge tube.
    • ii.
      Add 49.8 mL of culture-grade water to the tube and vortex to completely dissolve the NaOH.
    • iii.
      Filter the NaOH solution through a 0.22 μm filter into a new 50 mL sterile centrifuge tube.
  • b.
    Centrifuge the tube containing Triiodothyronine powder at 500 × g for 5 min.
  • c.
    Add 1 mL of DMSO to 25 mg of Triiodothyronine powder.
  • d.
    Vortex the solution to dissolve the Triiodothyronine. If the powder does not dissolve completely, sonication may be applied.
  • e.
    Dilute 4 μL of the 25 mg/mL Triiodothyronine solution into 996 μL of 0.1 M NaOH solution, and vortex to achieve a 100 μg/mL Triiodothyronine solution.
  • f.
    Dilute 10 μL of the 100 μg/mL Triiodothyronine solution to 990 μL of culture-grade water, and vortex to achieve a 1 μg/mL Triiodothyronine solution.
  • g.
    Dilute 10 μL of the 1 μg/mL Triiodothyronine solution to 990 μL of culture-grade water, and vortex to achieve a 10 ng/mL Triiodothyronine solution.
  • h.
    Store the Triiodothyronine stock solutions at −80°C for up to six months
• 15.
  Prepare heat inactivated FBS (Fetal Bovine Serum).

  • a.
    Thaw the FBS for 12–16 h at 4°C and invert the bottle to ensure thorough mixing.
  • b.
    Aliquot 40 mL of FBS to a 50 mL sterile centrifuge tube.
  • c.
    Pre-warm a water bath to 56°C.
  • d.
    Incubate the 40 mL FBS in the 56°C water bath for 30 min.
  • e.
    Store the heat-inactivated FBS at 4°C for up to one month.

Prepare reagents for RPE cell digestion

Timing: 1 h

• 16.
  Prepare Papain stock solution.

  • a.
    Add 25 mL of DPBS containing Ca²⁺/Mg²⁺ to 25 mg of Papain powder (15 U/mg), and gently swirl the bottle to completely dissolve the powder.
  • b.
    Filter the Papain solution through a 0.22 μm filter into a new 50 mL sterile centrifuge tube.
  • c.
    Aliquot 5 mL of the 15 U/mL Papain stock solution per tube into 15 mL sterile centrifuge tubes.
  • d.
    Store the stock solution at 4°C for up to six months.

Note: Calcium and magnesium ions help stabilize the structure of papain and are beneficial to its activity.⁹ Adding Ca²⁺ and Mg²⁺ to the papain stock solution helps maintain its activity during storage.

• 17.
  Prepare Papain activation solution.

  • a.
    Weigh 160.7 mg of EDTA powder and transfer it to a 50 mL sterile centrifuge tube.
  • b.
    Add 40 mL of DPBS and vortex to completely dissolve the EDTA.
  • c.
    Add 2.349 mL of 2-Mercaptoethanol to the EDTA solution.
  • d.
    Adjust the final volume to 50 mL with DPBS and mix by vortexing.
  • e.
    Filter the Papain activation solution through a 0.22 μm filter into a new 50 mL sterile centrifuge tube.
  • f.
    Store the Papain activation solution (11 mM EDTA and 0.67 mM 2-Mercaptoethanol) at 4°C for up to one month.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse Monoclonal antibody anti-Bestrophin-1 (1:1000 (v/v))	Proteintech	Cat#60326-1-Ig; RRID: AB_2881436
Rabbit Polyclonal antibody anti-ZO-1 (1:1000 (v/v))	Proteintech	Cat#21773-1-AP; RRID: AB_10733242
PDGFRB Recombinant Rabbit Monoclonal Antibody (206), FITC (1:200 (v/v))	Invitrogen	Cat#MA5-52221; RRID: AB_3248698
Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 (1:1000 (v/v))	Invitrogen	Cat#A32744; RRID: AB_2762826
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (1:1000 (v/v))	Invitrogen	Cat#A32790; RRID: AB_2762833
Chemicals, peptides, and recombinant proteins
mTeSR1	Stem Cell Technologies	Cat#85850
Nuwacell Vitronectin	Shownin	Cat#RP01002
Y-27632 dihydrochloride	TargetMol	T1725; CAS: 129830-38-2
DPBS	Gibco	Cat#C14190500BT
DPBS, calcium, magnesium	Gibco	Cat#14040133
EDTA	Shownin	Cat#RP01007
DMSO	Sigma-Aldrich	D8418; CAS: 67-68-5
Matrigel	Corning	Cat#354230
DMEM/F12	Gibco	Cat#C11330500BT
N-2 Supplement	Gibco	Cat#17502048
B27 (with Vitamin A)	Gibco	Cat#17504044
MEM Non-Essential Amino Acids Solution	Gibco	Cat#11140050
Nicotinamide	Sigma-Aldrich	N0636; CAS: 98-92-0
Recombinant Mouse Noggin Protein	R&D Systems	1967-NG; GenPept: P97466
Recombinant Human IGF-I/IGF-1 Protein	R&D Systems	291-G1; GenPept: P05019
Recombinant Human Dkk-1 Protein	R&D Systems	5439-DK; GenPept: O94907
Recombinant Human/Mouse/Rat Activin A Protein	R&D Systems	338-AC; GenPept: P08476
Human FGF-basic (FGF-2/bFGF) (154 aa) Recombinant Protein	PeproTech	Cat#100-18B
SU5402	Santa Cruz Biotechnology	sc-204308; CAS: 215543-92-3
CHIR99021	Sigma-Aldrich	SML1046; CAS: 252917-06-9
Fetal Bovine Serum	Vazyme	Cat#F101-01
GlutaMAX Supplement	Gibco	Cat#35050061
Sodium pyruvate	Gibco	Cat#11360070
Penicillin-Streptomycin	Gibco	Cat#15140122
BASIC MEM alpha	Gibco	Cat#C12571500BT
Taurine	Sigma-Aldrich	T8691; CAS: 107-35-7
Hydrocortisone	Selleck Chemicals	S1696; CAS: 50-23-7
Triiodothyronine	Selleck Chemicals	S5726; CAS: 6893-02-3
Papain	Worthington Biochemical	Cat#LS003126
StemPro Accutase	Gibco	Cat#A1110501
CryoStor CS10	Stem Cell Technologies	Cat#100-1061
4% Paraformaldehyde	Biosharp	Cat#BL539A
Triton X-100	Sigma-Aldrich	T8787; CAS: 9036-19-5
BSA	Sigma-Aldrich	V900933; CAS: 9048-46-8
DAPI	Sigma-Aldrich	D9542; CAS: 28718-90-3
Trypsin-EDTA (0.25%)	Gibco	Cat#C25200072
2-mercaptoethanol	Sigma-Aldrich	M3148; CAS: 60-24-2
TRIzol reagent	Invitrogen	Cat#15596026CN
Experimental models: Cell lines
Human: iPSC, Male, 29 years, healthy subject	Yunce Biotech	Cat#YC-010001
Software and algorithms
Salmon	Patro et al.10	https://github.com/COMBINE-lab/salmon
DESeq2	Love et al.11	https://github.com/thelovelab/DESeq2
ClusterProfiler	Xu et al.12	https://github.com/YuLab-SMU/clusterProfiler
Other
6-well plate	NEST	Cat#703001
12-well plate	NEST	Cat#712001
Glass bottom cell culture dish	NEST	Cat#801002
3 mL Pasteur pipette, extra-long	NEST	Cat#318314
40 μm cell strainer	Biosharp	Cat#BS-40-CS
0.22 μm PES membrane filter	Millipore Express	Cat#SLGPR33RB
2.0 mL Cryogenic Vials	Corning	Cat#430488
Confocal laser scanning microscope	Zeiss	LSM800
Flow cytometer	Beckman	CytoFLEX SRT
VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina	Vazyme	Cat#NR605-01

Materials and equipment

RDM Base Medium

Reagent	Final concentration	Amount
DMEM/F12	N/A	48 mL
N2 Supplement	1%	0.5 mL
B27 (with Vitamin A)	2%	1 mL
MEM Non-Essential Amino Acids Solution	1%	0.5 mL
Total	N/A	50 mL

Store RDM Base Medium at 4°C for up to 1 month. N/A = Not applicable.

D0-2 RDM

Reagent	Final concentration	Amount
RDM Base Medium	N/A	9.9 mL
NIC	10 mM	100 μL
Noggin	50 ng/mL	5 μL
DKK-1	10 ng/mL	1 μL
IGF-1	10 ng/mL	1 μL
Total	N/A	10 mL

Store D0-2 RDM at 4°C for up to 1 week. N/A = Not applicable.

D2-4 RDM

Reagent	Final concentration	Amount
RDM Base Medium	N/A	9.9 mL
NIC	10 mM	100 μL
Noggin	10 ng/mL	1 μL
bFGF	5 ng/mL	1 μL
DKK-1	10 ng/mL	1 μL
IGF-1	10 ng/mL	1 μL
Total	N/A	10 mL

Freshly prepare the D2-4 RDM before use. N/A = Not applicable.

D4-6 RDM

Reagent	Final concentration	Amount
RDM Base Medium	N/A	10 mL
Activin A	100 ng/mL	10 μL
DKK-1	10 ng/mL	1 μL
IGF-1	10 ng/mL	1 μL
Total	N/A	10 mL

Freshly prepare the D4-6 RDM before use. N/A = Not applicable.

D6-8 RDM

Reagent	Final concentration	Amount
RDM Base Medium	N/A	10 mL
Activin A	100 ng/mL	10 μL
SU5402	10 μM	10 μL
Total	N/A	10 mL

Freshly prepare the D6-8 RDM before use. N/A = Not applicable.

D8-16 RDM

Reagent	Final concentration	Amount
RDM Base Medium	N/A	10 mL
Activin A	100 ng/mL	10 μL
SU5402	10 μM	10 μL
CHIR99021	3 μM	10 μL
Total	N/A	10 mL

Store D8-16 RDM at 4°C for up to 1 week. N/A = Not applicable.

RPE Maintenance Medium

Reagent	Final concentration	Amount
DMEM/F12	N/A	46.5 mL
Heat inactivated FBS	2%	1 mL
GlutaMAX Supplement	1%	0.5 mL
MEM Non-Essential Amino Acids Solution	1%	0.5 mL
Sodium pyruvate	1%	0.5 mL
Penicillin-Streptomycin	1%	0.5 mL
Nicotinamide	10 mM	0.5 mL
Total	N/A	50 mL

Store RPE Maintenance Medium at 4°C for up to 1 month. N/A = Not applicable.

RPE Maturation Medium

Reagent	Final concentration	Amount
BASIC MEM alpha	N/A	45.135 mL
Heat inactivated FBS	5%	2.5 mL
N2 Supplement	1%	0.5 mL
MEM Non-Essential Amino Acids Solution	1%	0.5 mL
Sodium pyruvate	1%	0.5 mL
Penicillin-Streptomycin	1%	0.5 mL
Taurine	250 mg/L	0.25 mL
Hydrocortisone	20 μg/L	50 μL
Triiodothyronine	0.013 μg/L	65 μL
Total	N/A	50 mL

Store RPE Maturation Medium at 4°C for up to 1 month. N/A = Not applicable.

Step-by-step method details

iPSC culture and passage

Timing: 5–15 days

Timing: 1.5 h (step 1)

Timing: 3–7 days (step 2)

Timing: 2 h (step 3)

Timing: 1 h (step 4)

This section will describe the steps to thaw, culture, and passage iPSCs.

Note: Operate cells using a 3 mL Pasteur pipette.

• 1.
  Prepare Vitronectin-coated 6-well plate.

  • a.
    Thaw a vial of the pre-aliquoted Vitronectin (VTN, 0.5 mg/mL) at 4°C for 30 min.
  • b.
    Add 2 mL of DMEM/F12 medium to a well of a new 6-well plate.
  • c.
    Add 20 μL of VTN to the medium (to a final concentration of 5 μg/mL VTN).
  • d.
    Mix thoroughly by pipetting up and down using a 1 mL pipette.
  • e.
    Incubate the plate at 37°C for a minimum of 1 h.
• 2.
  Thaw and culture iPSCs.

  • a.
    Prewarm a water bath to 37°C. Bring the DMEM/F12 medium and mTeSR1 complete medium to 18–25°C from 4°C to prewarm.
  • b.
    Thaw a tube of frozen iPSCs (∼1×10⁶ cells) in the 37°C water bath with gentle swirling.
  • c.
    Transfer the thawed cells to a new 15 mL centrifuge tube using a 3 mL Pasteur pipette.
  • d.
    Slowly add 5 mL of DMEM/F12 medium to the 15 mL centrifuge tube and mix by gently inverting.
  • e.
    Centrifuge at 100 × g for 1 min.
  • f.
    Aspirate the supernatant and add 2 mL of mTeSR1 complete medium to the centrifuge tube.
  • g.
    Add 1 μL of Y-27632 stock solution into the centrifuge tube (to a final concentration of 5 μM Y-27632).
  • h.
    Gently resuspend the cell aggregates by pipetting up and down slowly a few times with a 3 mL Pasteur pipette.
  • i.
    Aspirate the medium from the VTN-coated well, and transfer cells into the VTN-coated well.
  • j.
    Gently shake to distribute the cells evenly, then incubate at 37°C with 5% CO₂.
  • k.
    Replace the medium daily with 2 mL of fresh mTeSR1 complete medium (without Y-27632).
  • l.
    Passage cells when they reach 80%–90% confluence.

Note: If cell confluence does not reach 80%–90% within one week after seeding, the cells should be passaged. Do not keep cells growing on a plate for more than one week.

• 3.
  Passage iPSCs.

  • a.
    Prepare VTN-coated 6-well plate as described in step 1.
  • b.
    Prewarm the DPBS and EDTA solution to 37°C in a water bath. Bring the mTeSR1 complete medium to 18–25°C from 4°C.
  • c.
    Aspirate mTeSR1 medium. Wash the cells with 2 mL of DPBS three times.
  • d.
    Add 2 mL 37°C prewarmed EDTA to each well of cells.
  • e.
    Incubate cells at 37°C for 8-10 min. Monitor the iPSCs under a phase-contrast microscope to ensure that 80%–90% of cells appear brighter.
  • f.
    Aspirate EDTA carefully, avoiding stripping cells from the plate.
  • g.
    Slowly add 2 mL of mTeSR1 complete medium.

    • i.
      Gently rock the plate to detach the cells.
    • ii.
      Check the iPSC aggregates under a phase-contrast microscope.
    • iii.
      To achieve aggregates containing 5–15 cells, drop the cells from a height of 3-5 cm by using a 3 mL Pasteur pipette. troubleshooting 1.
  • h.
    Aspirate the medium from the VTN-coated well and transfer 0.25 mL of cell suspension (1:8 dilution) to a well of a VTN-coated 6-well plate.
  • i.
    For each well, add 1.75 mL of fresh mTeSR1 complete medium and 1 μL of Y-27632 stock solution.
  • j.
    Shake the cells and place the plate in a 37°C incubator with 5% CO₂.
  • k.
    Replace the medium daily with 2 mL of fresh mTeSR1 complete medium (without Y-27632).

Note: Ensure that iPSCs remain in aggregate form. Avoid prolonged EDTA digestion, as extended exposure may cause dissociation of iPSCs into single cells.

• 4.
  Freezing iPSCs.

  • a.
    Digest iPSCs as described in step 3 b–f.
  • b.
    Mix 0.3 mL of cell culture grade DMSO with 0.7 mL of mTeSR1 complete medium.
  • c.
    Add the mixture to the cell suspension to achieve a total volume of 3 mL, resulting in a final concentration of 10% DMSO.
  • d.
    Mix the cell suspension by gently pipetting up and down with a 3 mL Pasteur pipette.
  • e.
    Aliquot 1 mL of the cell suspension into a cryopreservation tube.
  • f.
    Freeze cells by putting the tubes in a −80°C ultra-cold storage freezer with a programmable cryopreservation container.

Differentiation of RPE cells from iPSCs

Timing: 60 days

Timing: 2 h (step 5)

Timing: 1 h (step 6)

Timing: 2–3 h (step 13)

This section outlines the procedure for generating RPE cells from iPSCs (Figure 1).

Note: Differentiation can begin once iPSCs reach more than 80% confluence. Handle the cells using a 3 mL Pasteur pipette.

• 5.
  Prepare Matrigel-coated 12-well plate.

  • a.
    Thaw a vial of the pre-aliquoted Matrigel at 4°C for 30 min.
  • b.
    While the Matrigel is thawing, add 6 mL of fresh DMEM/F12 medium to a 15 mL centrifuge tube.
  • c.
    Place the centrifuge tube at −20°C for 30 min to pre-chill the medium.
  • d.
    Once the Matrigel has thawed and part of the medium has frozen, place both on ice and transfer them to the biosafety cabinet.
  • e.
    Pre-chill the tip by pipetting the pre-chilled medium up and down using a 100 μL pipette.
  • f.
    Add 100 μL of the Matrigel to the pre-chilled medium quickly (to a final concentration of 166 μg/mL Matrigel).
  • g.
    Vortex the medium for 2 min to mix and fully thaw any remaining ice.
  • h.
    Add 0.6 mL of medium to each well of a new 12-well plate, and incubate the plate at 37°C for at least 1 hour.
• 6.
  Day 0: start differentiation.

  • a.
    Prepare D0-2 RDM by adding 100 μL NIC, 5 μL Noggin, 1 μL DKK-1 and 1 μL IGF-1 to 10 mL of RDM Base Medium.
  • b.
    Aspirate mTeSR1 medium and wash cells three times with 2 mL of DPBS.
  • c.
    Add 2 mL prewarmed (37°C) EDTA to each well of cells.
  • d.
    Incubate cells at 37°C for 8–10 min. Monitor the iPSCs under a phase-contrast microscope to ensure that approximately 90% of cells appear brighter.
  • e.
    Aspirate EDTA carefully, avoiding detachment of the cells from the plate.
  • f.
    Slowly add 4 mL of D0-2 RDM to each well.

    • i.
      Gently rock the plate to detach the cells.
    • ii.
      Check the iPSC aggregates under a phase-contrast microscope.
    • iii.
      To obtain aggregates containing 5–15 cells, drop the cells from a height of 3–5 cm.
  • g.
    Aspirate the medium from the wells of the Matrigel-coated 12-well plate prepared in step 1.
  • h.
    Transfer the cell suspension to the Matrigel-coated 12-well plate.

    CRITICAL: Passage cells from one well of a 6-well plate (approximately 3 × 10ˆ6 cells) into four wells of the 12-well plate.

  • i.
    Shake the plate and place it in a 37°C incubator supplemented with 5% CO₂.

    CRITICAL: The confluence of iPSCs is crucial for successful differentiation. Low confluence results in an insufficient cell population, which can lead to significant cell death between day 4 to day 8 of differentiation (Figure 2). Additionally, when collecting cells by centrifugation, consider potential cell loss during the collection process. To minimize this, passage cells into fewer wells.

• 7.Day 1: Replace the medium with 1 mL of fresh D0-2 RDM.

CRITICAL: The proliferation of cells will be restrained during day 0-2.

Note: It is recommended to bring the culture medium to 18-25°C rather than 37°C before use.

• 8.Day 2: Prepare D2-4 RDM by adding 100 μL NIC, 1 μL Noggin,1 μL bFGF, 1 μL DKK-1 and 1 μL IGF-1 to 10 mL of RDM Base Medium. Replace the medium with 1 mL of fresh D2-4 RDM.
• 9.Day 4: Prepare D4-6 RDM by adding 10 μL Activin A, 1 μL DKK-1 and 1 μL IGF-1 to 10 mL of RDM Base Medium. Replace the medium with 1 mL of fresh D4-6 RDM.

Note: At this stage, cells should reach 100% confluence.

• 10.Day 6: Prepare D6-8 RDM by adding 10 μL Activin A and 10 μL SU5402 to 10 mL of RDM Base Medium. Replace medium with 1 mL of fresh D6-8 RDM.

Note: Significant cell death may occur during days 4-8 of differentiation. Insufficient initial cell density can result in failed differentiation. Troubleshooting 2.

• 11.Day 8-14: Prepare D8-16 RDM by adding 10 μL Activin A, 10 μL SU5402 and 10 μL CHIR99021 to 10 mL of RDM Base Medium. Replace the medium with 1 mL of fresh D8-16 RDM every 2 days.

Note: At this stage, the RPE progenitor sheets may contain non-RPE islands, and cells in these areas will gradually die. Troubleshooting 3.

• 12.Day 16-28: Replace the medium with 1 mL of fresh RPE Maintenance Medium every 2 days.

Note: At this stage, non-RPE cells will gradually undergo cell death, and later the gaps left by dead cells will be filled by proliferating cells.

• 13.
  Day 30: Freeze or passage RPE progenitor cells.

  • a.
    Add 50 μL of Papain activation solution to 450 μL of Papain stock solution. Mix thoroughly and incubate at 37°C for 30 min to activate the enzyme.
  • b.
    Aspirate RPE Maintenance Medium. Wash the cells three times with 1 mL of DPBS.
  • c.
    Add 0.5 mL of prewarmed Papain solution (15 U/mL, 37°C) to each well. Shake the plate to allow the enzyme solution to cover the cells evenly.
  • d.
    Incubate cells at 37°C for 30–60 min until the cells detach from the plate in large sheets.
  • e.
    Carefully remove the Papain using a 1 mL pipette without aspirating the cells.
  • f.
    Add 0.5 mL of prewarmed Accutase (37°C) to each well. Shake the plate to allow the enzyme solution to cover the cells evenly.
  • g.
    Incubate cells at 37°C for 30–60 min. Gently pipette the cells up and down with a 1 mL pipette to dissociate the sheet into single cells or small aggregates.
  • h.
    Transfer the cell suspension into a 15 mL centrifuge tube and centrifuge at 300 × g for 5 min. Aspirate the supernatant.
  • i.
    Add 3 mL of DPBS to wash the cells. Centrifuge again and aspirate the supernatant.
  • j.
    Proceed to freeze or passage the cells as required.

    • i.
      Freeze cells: Add 1 mL of CryoStor CS10 to the centrifuge tube containing the cell pellet.
    • ii.
      Gently pipette up and down to resuspend the cells.
    • iii.
      Aliquot the suspension into a cryopreservation tube.
    • iv.
      Passage cells: Add 4 mL of RPE Maturation Medium to the centrifuge tube and resuspend the cells.
    • v.
      Transfer the cell suspension to a Matrigel-coated 12-well plate.

CRITICAL: Passage cells from one well of a 12-well plate into four wells of a new 12-well plate (∼3.0 × 10ˆ5 cells per 12-well plate well).

Pause point: Frozen RPE progenitor cells can be stored in the vapor phase of liquid nitrogen for more than one year.

• 14.Day 30-60: Replace the medium with 1 mL of fresh RPE Maturation Medium every 3 days.

Note: Pigmentation typically begins to appear around day 40 (Figure 3).

Figure 1.

Differentiation of RPE cells from iPSCs

(A) Schematic diagram of the RPE differentiation protocol.

(B) Phase-contrast images illustrating morphological changes throughout the differentiation process. Scale bar: 150 μm.

Figure 2.

Impact of initial cell density on cell survival from Day 4 to Day 8

Increased initial cell density correlates with improved cell survival between day 4 to day 8 of differentiation. Scale bar: 300 μm.

Figure 3.

Cobblestone-like morphology and pigmentation of RPE cells at day 45

Phase-contrast image showing RPE cells exhibiting characteristic cobblestone-like morphology and pigment accumulation at day 45 of differentiation. Scale bar: 50 μm.

Identification of mature RPE cells by immunofluorescence

Timing: 2 days

This section describes the procedure for identifying mature RPE cells by immunofluorescence.

Note: In this protocol, cells were transferred to a Matrigel-coated glass-bottomed dish on day 30 for observation by confocal microscopy.

• 15.
  Cell Fixation.

  • a.
    Aspirate the culture medium and wash cells three times with 1 mL of DPBS.
  • b.
    Aspirate the DPBS and add 0.5 mL of 4% paraformaldehyde per well. Fix the cells for 20 min at 18–25°C.
  • c.
    Remove the paraformaldehyde and wash the cells three times with 1 mL of DPBS.
• 16.
  Immunofluorescent staining.

  • a.
    Prepare 0.1% PBST by adding 100 μL of Triton X-100 to 100 mL of DPBS.
  • b.
    Aspirate the DPBS and add 0.5 mL of 0.3% PBST (300 μL Triton X-100 in 100 mL DPBS). Incubate for 10 min at 18–25°C.
  • c.
    Aspirate the 0.3% PBST and add 0.5 mL of 3% BSA (3 g BSA in 100 mL of 0.1% PBST). Incubate for 30 min at 18–25°C.
  • d.
    Prepare primary antibody solution by adding both Bestrophin-1 mouse monoclonal antibody and ZO-1 rabbit polyclonal antibody to 3% BSA, each to a final dilution of 1:1000 (v/v).
  • e.
    Aspirate the 3% BSA and add 0.3 mL of primary antibody solution. Incubate for 12–16 h at 4°C.
  • f.
    Remove the primary antibody solution. Wash the cells three times with 1 mL of 0.1% PBST.
  • g.
    Prepare secondary antibody solution by adding both donkey anti-mouse IgG Alexa Fluor Plus 594 antibody and donkey anti-rabbit IgG Alexa Fluor Plus 488 antibody to 0.1% PBST, each to a final dilution of 1:1000 (v/v).
  • h.
    Aspirate the 0.1% PBST. Add 0.3 mL of secondary antibody solution and incubate in the dark for 1 hour at 18–25°C.
  • i.
    Remove the secondary antibody solution. Wash the cells three times with 1 mL of 0.1% PBST.
  • j.
    Aspirate the 0.1% PBST and add 0.5 mL of DAPI solution (1 μg/mL in 0.1% PBST). Incubate in the dark for 5 min at 18–25°C.
  • k.
    Aspirate the DAPI solution. Wash the cells three times with 1 mL of 0.1% PBST.
• 17.Capture fluorescence images using a confocal microscope. troubleshooting 4.

Note: Use the Z-axis imaging function to acquire Z-stacks. Cell polarity can be assessed by correlating ZO-1 fluorescence localization with cell position^13,14 (Figure 4).

Figure 4.

Immunofluorescence analysis of mature RPE cells differentiated from iPSCs

Representative immunofluorescence images of mature RPE cells obtained by (A) fluorescence microscopy (scale bar: 750 μm) and (B) confocal microscopy (scale bar: 40 μm). The basolateral membrane marker BEST1 (red) and the nuclear dye DAPI (blue) reveal cell morphology.^15,16 The tight junction marker ZO-1 (green), localized exclusively to the apical region of the cells, indicates the polarity of mature RPE cells.¹³

FACS-based purification of mature RPE cells by autofluorescence

Timing: 1 day

Timing: 3–4 h (step 18)

Timing: 15 min (step 19)

Timing: 2–4 h (step 20)

This section outlines the procedure for the purification of mature RPE cells by FACS, including cell processing and the flow sorting approaches.

• 18.
  Dissociate cells by sequential Papain and Accutase digestion.

  • a.
    Add 50 μL of Papain activation solution to 450 μL of Papain stock solution. Mix thoroughly and incubate at 37°C for 30 min to activate Papain.
  • b.
    Aspirate RPE Maturation Medium. Wash cells three times with 1 mL of DPBS.
  • c.
    Add 0.5 mL of prewarmed Papain (15 U/mL, 37°C) to each well and shake the plate to allow the enzyme solution to cover the cells evenly.
  • d.
    Incubate cells at 37°C for 45–80 min. The cells will detach from the plate in large sheets.
  • e.
    Carefully remove the Papain using a 1 mL pipette. Do not aspirate the cells.
  • f.
    Add 0.5 mL of prewarmed Accutase (37°C) to the cells, shaking the plate to allow the enzyme solution to cover the cells evenly.
  • g.
    Incubate cells at 37°C for 45–80 min. After incubation, gently pipette the cells up and down using a 1 mL pipette to dissociate the cell sheets into single cells or small aggregates. troubleshooting 5.
  • h.
    Transfer the dissociated cells into a 15 mL centrifuge tube. centrifuge the cells at 300 × g for 5 min at 4°C. Aspirate the supernatant.
  • i.
    Wash the cells with 3 mL of cold DPBS (4°C).
  • j.
    Filter the cell suspension through a 40 μm cell strainer and transfer to a 15 mL centrifuge tube. Centrifuge at 300 × g for 5 min at 4°C.
  • k.
    Aspirate the supernatant and resuspend the cells in 1 mL of cold DPBS (4°C).
  • l.
    keep the cells on ice, and cells are ready for FACS.

CRITICAL: By day 60, RPE cells develop a basement membrane-like structure to which they are firmly attached. Effective digestion of this membrane relies primarily on Papain; therefore, ensure complete Papain digestion before adding Accutase. (Figure 5).

Note: Both Papain and Accutase solutions maintain high cell viability, so prolonged digestion is not detrimental. Ensure digestion is thorough for optimal cell dissociation (Figure 6).

• 19.
  Prepare a control cell suspension. Use non-pigmented cells from one well of a 6-well plate as a control. In this protocol, the undifferentiated ARPE-19 cells were used as a control.

  • a.
    Aspirate medium and wash cells three times with 2 mL of DPBS.
  • b.
    Aspirate the DPBS and add 0.5 mL of 0.25% Trypsin-EDTA. Incubate at 37°C for 3 min.
  • c.
    Gently pipette the cells up and down using a 1 mL pipette to make a single cell suspension.
  • d.
    Add 1 mL of medium to neutralize the 0.25% Trypsin-EDTA. Transfer the cells to a 15 mL centrifuge tube and centrifuge at 300 × g for 5 min at 4°C.
  • e.
    Aspirate the supernatant and resuspend the ARPE-19 cells in 1 mL of cold DPBS (4°C).
  • f.
    Keep the cells on ice, and cells are ready for FACS analysis.

CRITICAL: Step 3 can be initiated toward the end of step 1f to minimize the time that cells remain in DPBS before sorting.

Note: Culture ARPE-19 in DMEM/F12 medium supplemented with 10% FBS (Fetal Bovine Serum).

• 20.
  Use a flow cytometer to sort cells. (We use a Beckman CytoFLEX SRT to sort cells) (Figure 7).

  • a.
    Begin by analyzing the control cells first.

    • i.
      Draw a gate (P1) in the FSC-A/SSC-A dot plot to select intact cells and exclude debris (FSC-A < 1.0 × 10ˆ5, SSC-A < 1.0 × 10ˆ5).

      Note: P1 typically comprised > 80% of total recorded events.

    • ii.
      Draw a gate (P2) on the R780-H distribution plot, including cells from the lowest R780-H signal up to the 95th percentile of fluorescence intensity.

      Note: Ensure that this gate retains >95% of the P1 population.

      CRITICAL: Doublets (typically ∼10% of total P1 events) were not excluded using the FSC-H/FSC-A dot plot. The mature RPE cells can form tight junctions with each other; therefore, a portion of mature RPE cells may remain as doublets or small aggregates.

  • b.
    Sort mature RPE cells.

    • i.
      Draw a gate (P1) in the FSC-A/SSC-A dot plot to select intact cells and exclude debris (FSC-A < 1.0 × 10ˆ5, SSC-A < 1.0 × 10ˆ5).

      Note: P1 typically comprised > 80% of total recorded events.

    • ii.
      Apply the P2 gate established in step a (retaining the lower 95% of events by R780-H intensity in the control) to the R780-H distribution plot.
    • iii.
      Gate the events with R780-H signal above the P2 upper boundary as P3.
    • iv.
      Before sorting, add 2 mL of fresh RPE Maturation Medium to the collection tubes.
    • v.
      Collect both P2 cells (as a negative control) and P3 cells.
  • c.
    Transfer the sorted cells to a 15 mL centrifuge tube and centrifuge at 300 × g for 5 min at 4°C.

    Note: After centrifugation, the pigment of P2 cells should be significantly lower than that of P3 cells (Figures 7B and 10B).

  • d.
    Aspirate the supernatant.
  • e.
    To maintain the culture, resuspend the cells in 1 mL of RPE Maturation Medium and seed onto a Matrigel-coated 12-well plate (∼3.0 × 10ˆ5 cells per 12-well plate well).
  • f.
    Shake the plate to distribute the cells evenly, then place the plate in a 37°C incubator supplemented with 5% CO₂. troubleshooting 6.

    Note: Ensure the flow cytometer is prepared before processing cells, and minimize the sorting duration to preserve cell viability.

Figure 5.

Sequential digestion of RPE cells using Papain and Accutase

Sequential treatment with Papain followed by Accutase results in optimal dissociation of RPE cells, compared with either enzyme used alone. Scale bar: 300 μm.

Figure 6.

Papain-Accutase digestion preserves RPE cell viability

(A and B) Macroscopic (A) and phase contrast (scale bar: 300 μm) (B) images of RPE cells digested with 0.25% Trypsin-EDTA (2.5 mg/mL Trypsin and 0.91 mM EDTA, 20 min) or sequential Papain-Accutase.

(C–E) Trypan blue staining revealed a lower cell death rate (C and D) and higher cell yield (E) of RPE cells digested with Papain-Accutase compared with Trypsin-EDTA. Data are represented as mean ± SEM; p = 0.0011 (D) and p < 0.0001 (E) by Student’s t-test. Scale bar (C): 300 μm.

(F) Reseeding of dissociated cells following digestion with Trypsin-EDTA or Papain-Accutase, showing improved attachment and morphology with Papain-Accutase treatment. Scale bar: 300 μm.

Figure 7.

FACS-based purification of mature RPE cells

(A) ARPE-19 cells were used as control and cells were collected from the P2 and P3 gates.

(B and C) Cells collected from the P3 gate exhibited significant pigmentation, indicating a mature RPE phenotype. Scale bar (C): 150 μm.

Figure 10.

Comparison of RPE cells sorted by the surface marker CD140b and autofluorescence

(A) Sorting of RPE cells using either the surface marker CD140b or autofluorescence.

(B) Representative cell pellets obtained after FACS.

(C) PCA and (D) correlation analysis of RNA-seq data from sorted cells.

(E) Differential gene expression analysis between CD140b-high and R780-high cells, showing that R780-high cells exhibit higher expression of mature RPE markers.

Expected outcomes

We have described a detailed method for differentiating and purifying RPE cells from iPSCs. The differentiated cells were validated by immunofluorescence and subsequently enriched by FACS. When differentiation is initiated from a single well of a 12-well plate, the expected yield at day 60 (before FACS) is approximately 11.2 ± 1.6 million cells (n = 3). During FACS, the proportion of positive cells is typically 51.9 ± 4.5% (n = 3). The positive population exhibits pronounced pigmentation, whereas the negative population appears largely non-pigmented. Post-sorting, the cells maintain high viability and can be readily reseeded. 61.0 ± 3.5% (n = 3) of cells adhere within 24 hours and continue to proliferate over subsequent days (Figure 8).

Figure 8.

Reseeding and proliferation of mature RPE cells following FACS

Sorted mature RPE cells retain the ability to attach and proliferate after reseeding, demonstrating their viability and functional capacity post-sorting. Scale bar: 300 μm.

RNA-seq analysis was performed to compare the positive and negative populations after sorting. The positive cells displayed higher expression levels of multiple RPE cell markers. The GO terms analysis of significantly upregulated genes revealed strong enrichment for biological processes and cellular components associated with RPE identity, supporting the effectiveness of our sorting strategy (Figure 9).

Figure 9.

RNA-seq analysis of FACS-sorted RPE cell subpopulation

(A) Cluster heatmap of two cell subpopulations, each represented with two biological replicates.

(B) R780-high cells expressed higher levels of RPE cell marker genes, including TYR, RGR, RLBP1, BEST1, and RPE65, which are critical for the functional properties of mature RPE cells.

(C) GO term enrichment analysis of differentially expressed genes indicates that R780-high cells are strongly associated with RPE-related functions, whereas R780-low cells display gene expression patterns more closely related to muscle cells.

The RNA of cells was extracted using TRIzol reagent, and RNA-seq libraries were constructed using the RNA-seq Library Prep Kit from Vazyme. The FASTQ-format reads were trimmed by Trim Galore, and the expression levels of transcripts were quantified using Salmon. The differentially expressed genes (DEGs) were determined using the DESeq2 package in R, and GO enrichment analysis of the DEGs was performed using the clusterProfiler package in R.

We further compared our approach with a previously published sorting method based on the surface marker CD140b.¹⁷ Cells sorted by our approach exhibited greater pigmentation. Correlation and PCA analysis demonstrated clear separation between the two sorting strategies. Differentially expressed gene analysis showed that cells enriched using our strategy expressed higher levels of mature RPE markers (RGR and BEST1) and lower levels of progenitor or early RPE markers (PAX6 and TMEFF2). These results indicate that our approach obtains more mature RPE cells (Figure 10).

Quantification and statistical analysis

Calculation of cell death rate and viable cells per well

The cell death rate and the number of viable cells per well are determined by quantifying the concentrations of dead and live cells using a Neubauer counting chamber with Trypan blue staining (Figure 6D and 6E).

$$celldeathrate=deadcellconcentration/(deadcellconcentration+livecellconcentration)$$

$$viablecellsperwell=livecellconcentration\times volumeofcellsuspension$$

Limitations

The RPE differentiation and purification protocol described here may not be universally applicable to iPSCs from other species (such as murine) or ESC lines. This protocol has only been validated with human iPSCs. We have not attempted to use human ESC lines or iPSCs from other species, and these cell lines may require optimization. Certain lines may not respond effectively to this approach, necessitating the use of an alternative protocol or selecting a different cell line.

The FACS strategy employed in this protocol relies on the intrinsic autofluorescence of mature RPE cells, which is likely attributable to the pigment production. Therefore, non-RPE cells capable of synthesizing pigment, such as melanocytes, may also be captured in the positive subpopulation.

Due to resource limitations, the characterization of FACS-purified cells in this study was primarily based on RNA-seq analysis. Functional assessments, such as transepithelial electrical resistance (TER) measurements and phagocytosis assays, were not performed. Further functional validation would be beneficial to more thoroughly validate and refine this purification protocol.

Troubleshooting

Problem 1

Some iPSCs may fail to detach after EDTA digestion. Meanwhile, the detached cell aggregates are too small, which will lead to a significant amount of cell loss. Section “iPSC culture and passage”, step 3g.

Potential solution

This issue may arise from passaging iPSCs too late. To avoid this, do not culture cells for more than one week without passaging. If this problem occurs, refrain from prolonging EDTA treatment and discard all undetached cells.

Problem 2

Significant cell death occurs between days 6–8. Section “differentiation of RPE cells from iPSCs”, step 10.

Massive cell death starts at day 6, leading to low cell density by day 8. With continuous cell loss, only a few isolated viable cell spots remain by day 16.

Potential solution

The initial cell density is crucial in addressing this issue. There are some reasons that may contribute to a low starting cell density.

• •Centrifuge: On day 0 (step 6), we use D0-2 RDM medium to directly resuspend the iPSCs, thus avoiding centrifugation for cell collection. Approaches that collect cells by centrifugation or by simple sedimentation result in significant loss of small aggregates, which negatively reduces the starting cell density.
• •Over-digestion: Over-digestion of iPSCs with EDTA at the onset of differentiation may result in overly small aggregates or a high percentage of single cells. These small aggregates and single cells have reduced survival rates between day 0 to 2, leading to a low starting cell density.

Problem 3

Significant cell death occurs between days 8–16. Section “differentiation of RPE cells from iPSCs”, step 11.

During days 8–16, some cells undergo an increase in volume followed by death. In severe cases, almost all cells exhibit this phenomenon, leading to differentiation failure.

Potential solution

• •Effectiveness of recombinant proteins: The reduced effectiveness of recombinant proteins, particularly Activin-A, caused by improper storage may contribute to this issue. To mitigate this, ensure all recombinant proteins are aliquoted and avoid repeated freezing and thawing.
• •Cell state: Different iPSC lines may exhibit variable responses to differentiation conditions. Consider switching to an alternative cell line if differentiation repeatedly fails. Additionally, the passage number of iPSCs may impact the differentiation outcomes. In our experience, iPSCs with a passage number greater than 30 tend to fail in differentiation.

Problem 4

Failure of BEST1 Immunofluorescence. Section “identification of mature RPE cells by immunofluorescence”, step 17.

Potential solution

The expression of the RPE marker BEST1 depends on the maturity of the cells. Verify that the RPE Maturation Medium is properly prepared. Additionally, consider extending the maturation period at step 14 if further maturation is required.

Problem 5

Failure to separate cells during digestion. Section “FACS-based purification of mature RPE cells by autofluorescence”, step 18g.

Some cells fail to detach from the plate after Accutase digestion, while some cells remain aggregated together even after prolonged digestion.

Potential solution

Extend the Papain digestion process. Adequate Papain digestion is critical. Incomplete Papain digestion may lead to this issue. After sufficient Papain digestion, cells should be completely detached. At this stage, gently pipetting up and down can help dissociate the cell sheets into smaller aggregates. It is important to note that the digestive actions of Papain and Accutase are distinct, and extending Accutase treatment will not compensate for incomplete Papain digestion.

Problem 6

Cells are contaminated by bacteria after FACS. Section “FACS-based purification of mature RPE cells by autofluorescence”, step 20f.

Potential solution

• •Perform FACS in a biosafety cabinet to maintain a sterile environment. Ensure the flow cytometer is thoroughly cleaned before sorting.
• •Following flow sorting, supplement the RPE Maturation Medium with twice the standard concentration of penicillin-streptomycin to prevent contamination.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hao Qian (qianhao@uestc.edu.cn).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Fengming Yang (yangfengming01@outlook.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The datasets generated during this study are available at BioProject: PRJCA061105 (https://ngdc.cncb.ac.cn/bioproject/).

Acknowledgments

This work was funded by the National Key R&D Program of China (2023YFC2506100); National Natural Science Foundation of China (82271276 to H.Q. and 12102086 to J.H.); Sichuan Science and Technology Program (2025NSFJQ0065 to H.Q.); Hubei Science and Technology Program (2025AFD252 to H.Z.); and the Key Program of Scientific Research, Anhui Provincial Department of Education (2025AHGXZK30579 to R.G.).

Author contributions

Methodology, F.Y. and R.L.; validation, F.Y., R.L., X.L., B.C., J.L., and R.G.; resources, Y.L., L.R., and H.Q.; funding acquisition, J.H., R.G., H.Z., and H.Q.; writing – original draft, F.Y.; writing – review and editing, B.L., J.H., H.Z., and H.Q.

Declaration of interests

H.Q., F.Y., J.H., R.L., and X.L. have a granted patent (Patent no. ZL 2024 1 1928440.3) related to this work. Y.L. and L.R. are employees of Chengdu Yunce Medical Biotechnology Co., Ltd.