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Enhancing ESC-derived Therapies for Age Related Disorders

Review of “Engineering Efficient Retinal Pigment Epithelium Differentiation from Human Pluripotent Stem Cells” from SCTM by Stuart P. Atkinson

With a steady increase in the aged population, has come a steady increase in the requirement for therapies to treat age-related diseases. Disruption of the retinal pigment epithelium (RPE) is linked to age-related degenerative eye disorders [1], and while adult or fetal RPE cells are scarce, the production of RPE/early neural retinal cells from human embryonic stem cells (hESCs) is deemed a viable option [2, 3]. However, there are still barriers to the translation of hESC-derived retinal cells to the clinical setting, and there is no consensus on the most efficient protocol, or the methods used to determine efficiency. This has led the group of Pete Coffey (UCL Institute of Ophthalmology, London, UK) to develop a simple and robust method of quantifying RPE production from hESCs using a novel, feeder-free differentiation protocol that uses single cell dissociation and density-optimized seeding, in a move towards efficient large scale production of RPE [4].

The group utilized a modified scanning device to assess and quantify pigmentation in cultures differentiating in vitro as a method of quantifying RPE production. This initially found that different hESC lines had variable differentiation capacities following expansion of dissected “clumps” of pigmented foci from spontaneously differentiating cultures. Furthermore, relative age (passage number) and seeding density of the hESCs was also a factor in RPE yield; high passage cells gave a reduced number of foci, while a higher seeding density gave improved RPE differentiation efficiency. High density culturing also had the advantage of being monetarily cheaper with regards to both hESC and medium usage. To control for size or number, the authors attempted differentiation of RPE under feeder-free conditions from clonally propagated hESCs [5] (enzymatic dissociation and growth of hESCs on Matrigel with mTeSR1), and found that high density hESCs survived, grew to confluence and upon differentiation gave higher numbers of pigmented foci (per cm2) than previous clump-based techniques. Indeed, increasing cell density to a maximum of 35,000 cells per cm2 led to the highest observed differentiation efficiency (See Figure).

At this stage, the authors took a step back to assess whether directed differentiation using small molecule inhibiters could enhance RPE yield. Dorsomorphin (dual SMAD inhibitor) promoted neuroectodermal differentiation (Pax6+, Six3+, Lhx2+, Nestin+), whilst downregulating genes involved in mesoderm, endoderm and trophectoderm differentiation, thus promoting subsequent RPE differentiation. Indeed, 10 days of Dorsomorphin treatment led to a threefold increase in the number of pigmented foci per cm2 over a range of passages for one of the hESC lines assessed. However, addition of Dorsomorphin to another hESC line under similar conditions mediated a reduction in pigmentation, and the appearance of cells with neural progenitor morphology, accompanied by the downregulation of RPE specific gene expression. Emergent cells expressed Chx10, which is suggestive of an early neural retina lineage.

The cheap, flexible, non-destructive, semi-automated measurement of the rate of hESC differentiation towards RPE used in this study provides a means to assess progress and to allow intervention, and moves towards the industrial scale up of differentiation that is required for the clinical application of hESC-derived RPE. While this study is of obvious use to the field, it also highlights an important concept to many differentiation studies; each cell line is likely to respond differently to a given protocol and tailor-made changes are likely to be required. This may impact negatively on the costs and times involved in any potential therapy. However, for non-pigmented cell types, this may mean the modification of cells to express a specific reporter gene construct.


  1. Ramsden CM, Powner MB, Carr AJ, et al. Stem cells in retinal regeneration: past, present and future. Development 2013;140:2576-2585.
  2. Osakada F, Ikeda H, Mandai M, et al. Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nature biotechnology 2008;26:215-224.
  3. Nakano T, Ando S, Takata N, et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 2012;10:771-785.
  4. Lane A, Philip LR, Ruban L, et al. Engineering Efficient Retinal Pigment Epithelium Differentiation From Human Pluripotent Stem Cells. Stem Cells Translational Medicine 2014;
  5. Maruotti J, Wahlin K, Gorrell D, et al. A simple and scalable process for the differentiation of retinal pigment epithelium from human pluripotent stem cells. Stem Cells Translational Medicine 2013;2:341-354.