Summary: A refined protocol for converting human embryonic stem cells into retinal pigment epithelium (RPE) cells offers a potentially safe and effective treatment approach for age-related macular degeneration.
Source: EPFL
Aging affects the eyes in many ways. For most people it means changing glasses or reduced sharpness of vision, but some age-related conditions are far more serious. Age-related macular degeneration (AMD) targets the macula—the central region of the retina responsible for detailed, high-acuity vision—leading to blurring and loss of central sight.
The retina is a layered, light-sensitive tissue dominated by photoreceptors—cones and rods—that detect light. Directly beneath these photoreceptors lies the retinal pigment epithelium (RPE), a single-cell-thick layer that absorbs stray light, clears cellular debris, and supports the health and function of photoreceptors.
Because the RPE nourishes and maintains photoreceptors, replacing damaged or aged RPE cells is a promising strategy to slow or halt AMD. Researchers aim to derive healthy RPE cells from human embryonic stem cells (hESCs) to create transplantable cell therapies.
Several differentiation protocols exist to produce RPE from stem cells, but key questions remain about how cells progress through transitional states and how long the process should take. Some methods require months; others nearly a year. Understanding the dynamic sequence of cellular changes during differentiation is essential to improve safety, shorten production time, and increase purity of the final RPE product.
Mixed cell populations
“None of the differentiation protocols proposed for clinical trials have been scrutinized over time at the single-cell level – we know they can make retinal pigment cells, but how cells evolve to that state remains a mystery,” says Dr Gioele La Manno, a researcher with EPFL’s Life Sciences Independent Research (ELISIR) program.
“The field has focused heavily on the final product, and sometimes overlooked the developmental path the cells take,” he adds. For clinical translation, it is critical to monitor the trajectory cells follow: the route to maturity can affect treatment safety, cell purity, and manufacturing efficiency.
Tracking stem cells as they grow into RPE cells
La Manno led a collaborative study with Professor Fredrik Lanner at the Karolinska Institute (Sweden) to profile a clinically oriented protocol for differentiating hESCs into RPE cells. Their analysis demonstrates that the protocol can support development of safe, efficient pluripotent stem cell-based therapies for AMD. The work appears on the cover of Stem Cell Reports.
Traditional assays such as quantitative PCR and bulk RNA sequencing measure average gene expression across large cell populations, potentially masking important differences between individual cells. “In mixed-cell populations, these measurements may obscure critical differences between individual cells that are important for knowing if the process is unfolding correctly,” says Alex Lederer, a doctoral student at EPFL and one of the study’s lead authors.
To resolve cellular heterogeneity and track transitions precisely, the team used single-cell RNA sequencing (scRNA-seq). This approach profiles the active genes in individual cells at specific time points during differentiation, enabling a detailed view of intermediate states and rare subpopulations.
Looking at intermediate states
Using scRNA-seq, the researchers followed the gene-expression trajectories of individual hESCs through a sixty-day differentiation protocol. This high-resolution mapping revealed transient states and allowed the team to refine the protocol to suppress unwanted non-RPE cell fates, increasing the purity of the RPE population.
Their analysis showed that cells pass through a sequence that mirrors early embryonic development. The culture acquired a rostral embryo patterning signature—an early neural development program that normally gives rise to the neural tube and sensory systems—before cells converged toward an RPE identity.
At intermediate stages the team identified an NCAM1-positive retinal progenitor population and assessed its potential. By understanding and controlling these intermediate states, they were able to minimize contaminant cell types and favor the intended RPE lineage.
Eye-to-eye: transplanting RPE cells in an animal model
A critical goal is to produce a clean, transplant-ready RPE population for implantation into patients’ retinas. To test this, the researchers transplanted their scRNA-seq–characterized cells into the subretinal space of two female New Zealand white albino rabbits, a commonly used large-eyed animal model for retinal studies. The procedures were performed with approval from the Northern Stockholm Animal Experimental Ethics Committee.
The transplanted cells not only retained their RPE identity but continued to mature in vivo, demonstrating that the protocol yields cells capable of further development after implantation. “Our work shows that the differentiation protocol can develop safe and efficient pluripotent stem cell-based therapies for age-related macular degeneration,” says Dr Fredrik Lanner, who is working to advance the protocol toward clinical use.
Other contributors
- Novo Nordisk A/S
- University of California San Francisco
About this genetics and vision research news
Author: Nik Papageorgiou
Source: EPFL
Contact: Nik Papageorgiou – EPFL
Image: The image is in the public domain
Original Research: Open access.
“Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation” by Gioele La Manno et al. Stem Cell Reports
Abstract
Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation
Highlights
- Single-cell transcriptional analysis benchmarks hESC-derived RPE differentiation against in vivo RPE cells
- NCAM1 identified as a cell-surface marker of multipotent neuroepithelial progenitors
- hESC-to-RPE differentiation proceeds through a divergence-convergence trajectory
- hESC-derived RPE cells continue to mature in vivo after subretinal injection into a rabbit model
Summary
Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) represent a promising cell source to treat age-related macular degeneration (AMD). Although multiple clinical studies are underway, detailed single-cell mapping of transient states during in vitro differentiation has been lacking.
In this study, single-cell transcriptomic profiling was applied to an hESC-RPE differentiation protocol designed for clinical translation. The protocol drives cultures through a temporary diversification that resembles early embryonic patterning—cells quickly acquire a rostral embryo signature before converging on the RPE lineage.
At intermediate stages the team identified an NCAM1-positive retinal progenitor population and confirmed the protocol’s capacity to suppress non-RPE fates. The method yields a highly pure RPE population capable of further maturation following subretinal transplantation in a large-eyed animal model.
These molecular and functional evaluations of hESC-RPE differentiation support ongoing efforts to develop safe, efficient pluripotent stem cell-based therapies for AMD.