Effektiv Udledning af retinale pigmentepitel Celler fra stamceller

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 they are widely considered to be one single functional unit. (For a review of all the diverse functions of the RPE see Strauss, 2005¹.) Death or dysfunction of retinal pigment epithelium cells can induce age-related macular degeneration (AMD), the leading cause of permanent vision loss in industrialized countries^2-4.

AMD is a multifactorial disease of RPE, photoreceptors, and the choroidal vasculature; risk factors are diverse and include combinations of environmental and genetic influences^5,6. Treatments for AMD are very limited, but one promising potential therapy is RPE cell replacement^7,8. While the outcomes have been mixed, the transplantation of RPE cells in AMD patients (and in other patients with retinal degeneration) and also in rodent models of retinal degeneration, has the potential to transiently prevent significant photoreceptor atrophy^9-23. (The animal model commonly used for these studies are Royal College of Surgeons (RCS) rats, which harbor a mutation in the MerTK gene. This mutation renders RPE cells incapable of phagocytosing photoreceptor outer segments and promotes retinal degeneration²⁴.) While the reported survival rates of implanted RPE in the subretinal space of RCS rats and mice vary greatly, there is potential for them to survive for several months or years^9,10,12,20.

RPE cells can be obtained in sufficient numbers for transplantation by deriving them from pluripotent stem cells^9-14,25-28. Several independent groups have demonstrated that these cells function in similar ways to their somatic counterparts, and long term studies suggest that they are safe upon implantation in rat and mouse disease models^{9,10,12,14,19,20,25,29-32}. The use of induced pluripotent stem cells instead of embryonic stem cells may be advantageous since ethical issues and immunological challenges associated with allogeneic RPE may be obviated^33,34. Another exciting application for iPS technology is disease modeling³⁵. The ability to interrogate large numbers of RPE cells derived from patients with RPE diseases could be invaluable for understanding their molecular bases. This type of study has been performed recently with Best disease patient RPE and could pave the way for similar studies of inherited maculopathies³⁶.

The derivation of RPE from stem cells is a relatively simple process and can be done entirely in xeno-free conditions. The simplest strategy is to derive monolayers of RPE cells spontaneously, however, the yield can be significantly improved using directed differentiation techniques. But these techniques involve the use of exogenous protein factors that can be expensive and often generated in bacteria or other non-human sources^10,12,37. In our studies we followed an established protocol¹⁰ that utilizes nicotinamide and Activin A, a signaling factor that has been shown to be sufficient for RPE specification³⁸. Here we will demonstrate that the small molecule IDE-1 can adequately replace Activin A, thus reducing costs and alleviating concerns associated with the use of recombinant proteins³⁹. Additionally, we utilize xeno-free serum replacement, and we culture the differentiating RPE cells on a synthetic xeno-free substrate. RPE cells have been shown previously to differentiate very effectively using this approach⁴⁰.

When differentiated as a monolayer, we visualize pigmented colonies containing RPE cells after as early as five weeks¹². Once they reach sufficient size, they can be manually excised and transferred to another dish for expansion. RPE cells are notorious for dedifferentiating with each passage, and the use of anything older than five passages should be avoided (we find that sufficient numbers of cells for characterization and transplantation in animal models can easily be generated after two or three passages). Once fully differentiated, we employ multiple techniques to characterize the cells anatomically and functionally to ensure that they will serve as adequate replacements for diseased RPE. The description of these techniques, and protocol for implanting the iPS-RPE in the subretinal space of rodents, are beyond the scope of this methods paper and have been previously published^12,32,41.

While developing standardized protocols for effective derivation of iPS-RPE is clearly important for the clinics, there is also significant preclinical work to still be done in animal models. There are concerns regarding immunogenicity of iPS-derived cells, and multiple different implantation techniques, including implanting cells on artificial substrates, are being explored^42,43. For these reasons, we feel that the publication of standardized protocols is beneficial to facilitate both clinical and preclinical studies. Especially if direct comparisons will be done of iPS-RPE cells derived in different labs by different research groups.

Protocol

1. Direkte Differentiering af stamceller afledt RPE BEMÆRK: Alle inkubationstrin udføres ved 37 ° C i 5% CO 2 Opretholde HES eller hofter linjer (foder dagligt) i vedligeholdelse medier (MM, tabel 1). Når linjerne har nået tilstrækkelige standarder for kvalitetskontrol, holde dem på et lag af muse embryonale feeder celler (MEF) podet med en tæthed på 250.000 / brønd i en seks brønds plade. Xeno-fri kulturer kan ogs?…

Representative Results

Trinene i dette manuskript, som afbildet i figur 1, kan anvendes til let at generere RPE fra stamceller som tidligere rapporteret 10,12. Efter opretholdelse IPS linjer for flere uger, pigmenterede kolonier begynde at dukke op i de kolonier efter 5-7 uger (7 uger gamle kulturer er vist i figur 2A-C). Disse kolonier kan fortsætte med at vokse i uger kulturerne opretholdes. Når nå tilstrækkelige størrelser, som vist i figur 2D-F (8 uger gamle kulturer), kan…

Discussion

I dette manuskript vi skitsere de skridt til effektivt at generere et stort antal rene iPS-RPE kulturer. Vi har tidligere vist at bruge denne rettet differentiering protokol med Activin A, at vi kan generere iPS-RPE som stærkt ligner hRPE baseret på transcriptomics, proteomics, metabolomik og funktionalitet, og at de retard retinal degeneration, når implanteret i RCS rotter ^12,31,32 . Processen med at frembringe iPS-RPE er tidskrævende, men ikke besværlig (figur 1). Når IPS kulturer fly…

Materials

Name of Material/ Equipment (A-Z)	Company	Catalog Number	Comments/Description
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 ug 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 ug 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