Name: efficient derivation of retinal pigment epithelium cells from stem cells
Uploaded: 2015-03-08T14:00:00
Description: Watch this Scientific Journal Video about Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells at JoVE.com

We wish to thank the following individuals: Drs. Tim Krohne and Eyal Banin (along with Dr. Mandy Lehmann and David Friedlander) for generous help developing the differentiation protocols. Dr. Felicitas Bucher provided assistance differentiating the RPE cells used in this study. We also acknowledge the National Eye Institute (NEI grants EY11254 and EY021416), California Institute for Regenerative Medicine (CIRM grant TR1-01219), and the Lowy Medical Research Institute (LMRI) for very generous funding for this project.

1. Geregisseerd Differentiatie van stamcellen afgeleide RPE Opmerking: Alle incubatie stappen worden uitgevoerd bij 37 ° C in 5% CO2 <ol style="margin-left: 40px;"><li> Handhaving van de HES of heupen lijnen (dagelijks voeden) in onderhoud media (MM; tabel 1). Zodra de lijnen voldoende kwaliteits- controle bereikt, houden ze op een laag van muis embryonale voedingscellen (MEF) geënt bij een dichtheid van 250.000 / putje van een zes puts plaat. Xeno-vrij kweken...

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In dit manuscript schetsen we de stappen om efficiënt genereren van grote aantallen pure iPS-RPE culturen. We hebben eerder aangegeven met dit gericht differentiatie protocol met Activin A dat we iPS-RPE die sterk lijken hRPE op basis van transcriptomics, proteomics, metabolomics en functionaliteit kan genereren, en dat ze vertragen retinale degeneratie wanneer geïmplanteerd in RCS ratten 12,31,32 . Het maken van iPS-RPE is tijdrovend, maar niet bewerkelijk (figuur 1). Zodra de iPS kweken c...

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 countries2-4. 
AMD is a multifactorial disease of RPE, photoreceptors, and the choroidal vasculature; risk factors are diverse and include combinations of environmental and genetic influences5,6. Treatments for AMD are very limited, but one promising potential therapy is RPE cell replacement7,8. 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 atrophy9-23. (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 years9,10,12,20. 
RPE cells can be obtained in sufficient numbers for transplantation by deriving them from pluripotent stem cells9-14,25-28. 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 models9,10,12,14,19,20,25,29-32. 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 obviated33,34. 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 sources10,12,37. 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 published12,32,41. 
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 explored42,43. 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.

<table border="0" cellpadding="0" cellspacing="0" width="849">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Name of Material/ Equipment (A-Z)</td>
			<td style="width:145px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Corneal knife&#160;</td>
			<td style="width:145px;">Surgipro</td>
			<td style="width:119px;">SPOI-070</td>
			<td>knife x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">DMEM/F-12, HEPES</td>
			<td style="width:145px;">Life Technologies</td>
			<td>11330-032</td>
			<td>500 mL x 4&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate-Buffered Saline, 1X w/out Ca or Mg</td>
			<td style="width:145px;">VWR</td>
			<td style="width:119px;">45000-434</td>
			<td>500 mL x 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Fetal Bovine Serum, Regular (Heat Inactivated)</td>
			<td style="width:145px;">VWR</td>
			<td style="width:119px;">45000-736</td>
			<td>500 mL x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">FGF-Basic (AA 10-155) Recombinant Human Protein</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">PHG0021</td>
			<td>100 ug x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">IDE-1</td>
			<td style="width:145px;">Stemgent</td>
			<td style="width:119px;">04-0026</td>
			<td>2 mg x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Knockout DMEM</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">10829-018</td>
			<td>500 mL x 1&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">KnockOut Serum Replacement</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td>500 mL x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">L-Glutamine 200 mM&#160;</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td>100 mL x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution 100X&#160;</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td>100 mL x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Nicotinamide</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:119px;">N0636-100G</td>
			<td>100 g x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">15140-148</td>
			<td>20 mL x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Recombinant Human/Murine/Rat Activin A&#160;</td>
			<td style="width:145px;">PeproTech</td>
			<td style="width:119px;">120-14E</td>
			<td>10 ug x 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Synthemax-T Surface 6 Well Plates</td>
			<td style="width:145px;">Corning</td>
			<td style="width:119px;">3877</td>
			<td>Case(12) x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">TrypLE-Express Enzyme (1X), no phenol red&#160;</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">12604-021</td>
			<td>500 mL x 1&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Vacuum Filter/Storage Bottle System, 0.1&#181;m pore, 500mL&#160;</td>
			<td style="width:145px;">Corning</td>
			<td style="width:119px;">431475</td>
			<td>Case(12) x 1&#160;</td>
		</tr>
	</tbody>
</table>

efficient derivation of retinal pigment epithelium cells from stem cells

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.

De stappen in dit manuscript, zoals in figuur 1, kan worden gebruikt om gemakkelijk genereren RPE uit stamcellen zoals eerder gemeld 10,12. Na handhaven van de iPS lijnen enkele weken gepigmenteerde kolonies beginnen te verschijnen in de kolonies na 5-7 weken (7 weken oude kweken zijn weergegeven in Figuur 2A-C). Deze kolonies kunnen blijven groeien weken als de culturen blijven. Zodra bereiken voldoende afmetingen, zie figuur 2D-F (8 weken oude culturen), ku...

Watch this Scientific Journal Video about Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells at JoVE.com

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

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.

Efficiënte Afleiding van retinale pigment epitheel cellen uit stamcellen

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 ...

Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Afleiding van Cardiac stamcellen uit embryonale stamcellen

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

Afleiden van retinale pigment epitheel (RPE) van induced pluripotent stem (iPS) cellen door verschillende maten van embryoiden Bodies

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Feeder-vrij Afleiding van melanocyten uit menselijke pluripotente stamcellen

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Horizontale gehele berging: Een nieuw verwerkings- en beeldprotocol voor dikke, driedimensionale weefsel dwarsdoorsneden van de huid

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Snelle, gerichte differentiatie van de epitheliale cellen van de retinale Pigment van menselijke embryonale of geïnduceerde pluripotente stamcellen

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Eencellige Photoconversion in levende Intact zebravis

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

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Computationele analyse van de Caenorhabditis elegans Germline te bestuderen van de distributie van kernen, eiwitten en het cytoskelet

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Afleiding en differentiatie van Canine ovariële mesenchymale stamcellen

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Transplantatie-geschikt retinale Pigment epitheel Tissue Engineering afgeleid van menselijke embryonale stamcellen

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Efficiënt en schaalbare gestuurde differentiatie van klinisch hoornvlies Limbal epitheliale cellen van de stam van menselijke pluripotente stamcellen

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...