Table 2 Summary of methods for differentiation of human-induced pluripotent stem cells (hiPSC) to RPE

[11]

hiPSC

RPE

Direct differentiation; LDN193189, A-83–01, IWR-1-endo, Y-27632, CHIR99021, SU5402

34–36-day follow-up

•   Real-time PCR

•   Immunocytochemistry

•   Transepithelial electrical resistance

•   Phagocytosis assay

•   F-actin labeled imaging

RPE sheet

NA

•   NA

•   Obtaining high-purity RPE cells and mature RPE sheets without special selection

•   An automated, noninvasive TER prediction model based on F-actin-labeled images is developed to identify RPE sheets with low TER

•   Sample size limitation for model training

•   The limited reliability of prediction models; because each manufacturer must demonstrate its own manufacturing process, safety, and efficacy of its cellular products

[12]

hiPSC

RPE

Direct differentiation;

Noggin, IGF1, nicotinamide, Dkk1, bFGF, activin A, SU5402, VIP

24-day follow-up

•   Immunocytochemistry

•   Co-culture system

•   Flow cytometry

•   TUNEL assay

•   3D spheroid culture to culture spheroid RPE cells

•   Viability assay of spheroid RPE cells

•   Transepithelial electrical resistance

RPE cell suspension

Retinal degeneration 10 (rd10 mice)

•   Enzyme-linked immunosorbent assay

•   Hematoxylin and eosin staining

•   Western blots

•   Light avoidance behavior testing

•   Electroretinography

•   No evidence of rejection or tumorigenesis after subretinal injection for at least 2 weeks after transplantation

•   Co-culturing RPE by retinal explant or RGC confirmed the neuroprotective effect of secreted growth factors for retinal cells and retinal homeostasis

•   2 weeks is a rather short observation period postoperatively

[42]

hiPSCs

RPE

Spontaneous differentiation;

Removal of bFGF from the medium

60–90 days up to 8 months for different cell line follow-up

•   Morphological assessment

•   Quantitative real-time PCR

•   Immunocytochemistry

•   Immunoblot analysis

•   ROS phagocytosis

NA

NA

•   NA

•   Increases RPE65 protein expression

•   Different iPSC lines exhibit different propensities to spontaneously produce RPE

[46]

hiPSCs and retinal disease-specific hiPSCs

RPE sheet

Direct differentiation;

Noggin, bFGF, retinoic acid, and Shh

40-day follow-up

•   Flow cytometry

•   Immunofluorescence

•   Real-time PCR

NA

NA

•   NA

•   Differentiation of retinal disease-specific hiPSCs toward RPE; however, it was lower in comparison with normal hiPSCs

•   Additional assays such as quantitative ROS, phagocytosis, transepithelial resistance measurements, enzyme-linked immunosorbent and retinoid metabolism is also necessary for additional characterization of RPE

[47]

hESC or hiPSC

RPE

Direct differentiation;

Y-27632 ROCK inhibitor, LDN/SB, SB-431542, BMP 4/7, activin A based on adherent monolayer

Culture using xeno-free conditions

45-day follow-up

•   Real-time PCR

•   Enzyme-linked immunosorbent assay

•   Bead phagocytosis assay

•   Microarray Analysis

•   Immunocytochemistry

NA

NA

•   NA

•   Efficiently direct differentiation of pluripotent stem cells toward retinal pigment epithelium fate by using a simple culture

Stepwise modulation of activin A and BMP signaling method

•   Further work comparing the function of RPE derived from spontaneous and directed differentiation in an in vivo setting is needed

[48]

hiPSC

RPE

Direct differentiation;

Chetomin with nicotinamide

30-day follow-up

•   Real-time PCR

•   Flow cytometry

•   Immunostaining

•   Photoreceptor outer segment phagocytosis

•   VEGF and PEDF ELISA

Cell suspension

NOD-SCID mice

•   Fundus photographs

•   Immunostaining (2 weeks post-transplantation)

•   A high-throughput quantitative PCR screen was combined with a new RPE reporter assay based on hiPSCs to strongly induce the conversion of over half of the differentiating cells into RPE

•   A single passage of the whole culture produced a highly pure RPE cell population with many of the morphological, molecular, and functional characteristics of native RPE

•   There were no pigmented colonies when cultures were maintained in default medium during chetomin or chetomin/nicotinamide, suggesting that CTM-committed RPE cells are not fully mature in DM and require RPEM in order to attain their characteristic morphology

[49]

Oncogene mutation-free clinical-grade AMD patients-hiPSC

Clinical-grade RPE patches on PLGA scaffolds

Direct differentiation;

LDN-193189, SB431452, CKI-7 hydrochloride, IGF-1, PD0325901, nicotinamide, activin A

6-week follow-up for cell suspension and 10-week follow-up for patch RPE

•   Real-time PCR

•   Trans-epithelial resistance

•   Hexagonality measurement

•   Phagocytosis of photoreceptor outer segments

•   Lactic acid measurements

Cell suspension or patch (on PLGA Scaffold)

RCS rat

Pigs with laser-induced RPE injury

•   Optokinetic tracking

•   Optical coherence tomography and fluorescein angiography

•   Multi-focal Electroretinography

•   Immunostaining

•   A biodegradable PLGA scaffold approach improved integration and functionality of clinical-grade RPE patch in rats and porcines

•   No complete dose–response study was performed

•   An extensive set of reagents and quality control measures are required to ensure process consistency and reproducibility, which may increase the cost of manufacturing autologous cell therapy on a commercial scale

[51]

hiPSC

Retinal cells

Direct differentiation;

Noggin, Dkk-1, IGF-1, nicotinamide, FGF2, activin A, SU5402, CHIR99021, ROCK inhibitor

14-day follow-up

•   Morphology

•   Immunocytochemistry

Cell suspension

Pde6b knockout rats and SD rats

•   10-month follow-up

•   OCT imaging

•   ERG recording

•   Conventional PCR for validation of human mitochondrial DNA and Sanger sequencing

•   Hematoxylin and eosin staining

•   Immunohistochemical

•   Transplanted human iPSC-derived retinal cells exhibited characteristics of both RPE cells and photoreceptors

•   No abnormal cell proliferation nor morphological changes were observed in the subretinal space

•   After transplantation, the number of cells gradually decreased

•   The study did not reveal the presence of a network of retinal nerve cells in the region of transplantation or the linear stratification of mature retinal cells

[57]

hiPSC or

hESC

RPE

Direct Differentiation;

ROCK inhibitor (Y-27632), activin A

More than 5-week follow-up

•   Flow cytometry

•   Immunofluorescence

•   Hematoxylin–eosin staining

•   Immunostaining

•   Phagocytosis assay

•   Enzyme-linked immunosorbent assay

•   Transepithelial electrical resistance measurements

•   Scanning electron microscopy

•   Transmission electron microscopy

•   Single-cell RNA-sequencing bioinformatic analysis

Cell suspension

White albino rabbits

•   Bright-field imaging

•   Immunofluorescence

•   Identifying cell surface markers for RPE cells that can be used to develop a robust, direct, and scalable monolayer differentiation protocol as well as RPE cells isolation during in vitro differentiation with high quality and efficiency

•   An extensive analysis would be useful to determine whether the presence of eye-field progenitors in cell suspension would be beneficial or detrimental to functional integration following subretinal transplantation

•   The function of RPE cells in the retina must be confirmed after the transplantation of RPE cells

[59]

hiPSC

RPE monolayer

Activin A + simplified 2D culture in combination with lipoprotein uptake-based sorting (called the PLUS protocol)

90-day follow-up

•   Immunocytochemistry on cyst cryosections

•   Phagocytosis assay

•   AcLDL uptake assay

•   Real-time PCR analyses

•   Electron microscopy analyses

•   Enzyme-linked immunosorbent assay (ELISA) for vascular endothelial growth factor (VEGF)

•   Transepithelial electrical resistance measurements

•   Fluorescence-activated cell sorting of Dil-AcLDL positive cells

NA

NA

•   NA

•   This protocol obviates the need for growth factors and small chemical molecules and also is cost-effective

•   To ensure the safety of these RPE cells in clinical settings, it is vital to consider the implications of transplanting trace amounts of DiI-AcLDL as well as the safety of carbocyanine dyes alone or in conjunction with lipoproteins

[61]

hiPSC

RPE cell sheet on Soy Scaffold

Direct differentiation;

ROCK inhibitor

(Y-27632)

5–25-week follow-up

•   Immunohistochemistry

•   Enzyme-linked immunosorbent assay

•   RNA-seq analysis

NA

NA

•   NA

•   By cultivating RPE differentiated from hiPSCs on nanofibrous biomaterial scaffolds, whether synthetic or natural, a uniform expression of RPE maturation genes can be achieved

•   To evaluate the quality of differentiation on various substrates, RNA sequencing was applied

•   There is a need for a variety of assays, including structural, molecular, and physiological characteristics

[63]

hiPSC

and best disease patient-iPSCs

RPE monolayer

Direct differentiation;

OTX2, PAX6, and MITF transcription factors + Y27632, LDN-193189, SB-431542

At least 60-day follow-up

•   Real-time PCR

•   Immunocytochemistry

•   Flow cytometry

•   Phagocytosis with bovine rod outer segments

•   Western blotting

•   Bulk RNA-sequencing

•   RNA-seq data processing and analysis of differentially expressed genes

•   Automated 96 well plate imaging and analysis

NA

NA

•   NA

•   A high-efficiency and easily scalable differentiation strategy for generating iPSC-RPE from multiple patients and two wild-type iPSC lines by inducing the expression of OTX2, PAX6 and MITF (hOPM) by doxycycline paired with a small molecule

•   It is more appropriate to optimize differentiation requirements based on the cell line rather than the mutation in the disease

•   Aside from RPE differentiation induced by specialized media, neural retina neurons are also produced. In this case, it was necessary to express hOPM in order to drive the majority of iPSCs into the RPE lineage which could result in tumorigenesis or mutation; therefore, there is a need to monitor for either of these factors

[66]

HiPSC and patient’s specific iPSC

RPE

Spontaneous differentiation; xeno-free XVIVO-10 medium

without basic fibroblast growth factor

90-day follow-up

Direct differentiation; nicotinamide, noggin, Dkk1, IGF1, activin A, SU5402, and CHIR99021

14-day follow-up

•   Nicotinamide, noggin, Dkk1, IGF1, activin A, SU5402, and CHIR99021

•   Real-time quantitative polymerase chain reaction

•   Next-generation sequencing (RNA-seq)

•   Immunocytochemistry

•   Rod outer segment phagocytosis assay

•   Pigment epithelium-derived factor enzyme-linked immunosorbent assay

NA

NA

•   NA

•   Directed differentiation is a more reliable method for differentiating RPE from various pluripotent sources, and some iPSC lines are more capable of RPE differentiation. Extended culture times are needed for a fully mature RPE

•   It may be necessary to use directed methods rather than longer, spontaneous methods in some iPSC lines in order to [1] produce enough cells for characterization and [2] silence residual somatic cell lineage makers, both of which may require directed approaches over longer, spontaneous methods

[60]

hiPSC

RPE

Direct differentiation;

PD0325901, PD173074, Gö6983, LDN-193189, CHIR99021, SB431542, SAG, SU5402, CKI-7 and Fasudil was substituted for Y-27632

30-day follow-up

•   RT-PCR analysis

•   Immunofluorescence

•   Purity assay

•   Enzyme-linked immunosorbent assay (ELISA) for pigment epithelium-derived factor (PEDF)

•   Transepithelial electrical resistance measurement

Phagocytosis assay

NA

NA

•   NA

•   During the hiPSC maintenance period, transient inhibition of the FGF/MAPK pathway promotes functional RPE differentiation and eliminates the need for subsequent treatment with WNT and nodal signal inhibitors

Further inhibition of PKC or BMP signal increased differentiation efficiency

•   To reduce safety risks, such as product contamination, and to reduce manufacturing costs, the number of compounds in a drug formulation should be as small as possible

•   To maximize yield, the culture conditions need to be optimized to maximize target cell differentiation