Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Summary

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

Abstract

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Introduction

The various cell types that occupy the retina are organized in a functional architecture. The photoreceptors in the back of the retina are responsible for converting light into electrical impulses through phototransduction. However, phototransduction cannot occur without the coordinated efforts of other neighboring cell types including Mueller glia and retinal pigment epithelium (RPE) cells. A monolayer of RPE cells partitions the sensory retina from the choriocapillaris, the primary blood supply for photoreceptors, and are ideally situated to control multiple functions important for photoreceptor homeostasis. In fact, the RPE and photoreceptors are so co-dependent t....

Protocol

1. Directed Differentiation of Stem Cell-derived RPE

NOTE: All incubation steps are carried out at 37 °C in 5% CO₂

1. Maintain the hES or hiPS lines (feed daily) in maintenance media (MM; Table 1). Once the lines have reached sufficient standards of quality control, maintain them on a layer of mouse embryonic feeder cells (MEFs) seeded at a density of 250,000/well of a six well plate. Xeno-free cultures can also be generated following the protocol established by Tucker et al.⁴⁰.
2. Allow the stem cell colonies to reach confluency.
3. Switc....

Results

The steps outlined in this manuscript, as depicted in Figure 1, can be used to readily generate RPE from stem cells as previously reported^10,12. After maintaining the iPS lines for several weeks, pigmented colonies begin to appear in the colonies after 5-7 weeks (7 week old cultures are shown in Figure 2A-C). These colonies can continue to grow for weeks as the cultures are maintained. Once reaching sufficient sizes, as shown in Figure 2D-F (8 week old cultur.......

Discussion

In this manuscript we outline the steps to efficiently generate large numbers of pure iPS-RPE cultures. We have shown previously using this directed differentiation protocol with Activin A that we can generate iPS-RPE that strongly resemble hRPE based on transcriptomics, proteomics, metabolomics, and functionality, and that they retard retinal degeneration when implanted in RCS rats^12,31,32. The process of generating iPS-RPE is time-consuming, but not laborious (Figure 1). Once the iPS culture.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Corneal knife	Surgipro	SPOI-070	knife x 1
DMEM/F-12, HEPES	Life Technologies	11330-032	500 ml x 4
Dulbecco's Phosphate-Buffered Saline, 1X w/out Ca or Mg	VWR	45000-434	500 ml x 6
Fetal Bovine Serum, Regular (Heat Inactivated)	VWR	45000-736	500 ml x 1
FGF-Basic (AA 10-155) Recombinant Human Protein	Life Technologies	PHG0021	100 µg x 1
IDE-1	Stemgent	04-0026	2 mg x 1
Knockout DMEM	Life Technologies	10829-018	500 ml x 1
KnockOut Serum Replacement	Life Technologies	10828-028	500 ml x 1
L-Glutamine 200 mM	Life Technologies	25030-081	100 ml x 1
MEM Non-Essential Amino Acids Solution 100X	Life Technologies	11140-050	100 ml x 1
Nicotinamide	Sigma-Aldrich	N0636-100G	100 g x 1
Penicillin-Streptomycin (10,000 U/ml)	Life Technologies	15140-148	20 ml x 1
Recombinant Human/Murine/Rat Activin A	PeproTech	120-14E	10 µg x 2
Synthemax-T Surface 6 Well Plates	Corning	3877	Case(12) x 1
TrypLE-Express Enzyme (1X), no phenol red	Life Technologies	12604-021	500 ml x 1
Vacuum Filter/Storage Bottle System, 0.1µm pore, 500ml	Corning	431475	Case(12) x 1