Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model

Abstract

SummaryHuman embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.

Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage

“…An approach that is currently under substantial investigation is the replacement of dysfunctional, diseased RPE with cells derived in vitro from stem cell differentiation. Preclinical as well as early clinical reports suggest that this strategy is safe and allows the newly transplanted RPE to effectively perform functions such as phagocytosis, fluid and nutrient transport, and photoreceptor support, leading to potential restoration of vision . In addition, there are many features that make the eye an attractive organ for cell replacement and regenerative therapy approaches, such as the requirement of fewer cells for therapeutic intervention in comparison with other larger organs, accessibility and ease of monitoring, potential immune privilege, and presence of the retinal‐blood barrier, leading to separation from systemic circulation .…”

Human Fibrinogen for Maintenance and Differentiation of Induced Pluripotent Stem Cells in Two Dimensions and Three Dimensions

“…In efforts to evolve from laminin rich mouse ECM (Matrigel and Geltrex), most groups have focused on human laminin and vitronectin. Multiple publications have shown the ability to use these for culture of ESCs , and for laminins, iPSC culture. For example, Synthemax (Corning), a synthetic peptide with vitronectin elements, is a GMP‐compatible product marketed for ESC culture and which has seen limited use in the generation of iPSCs .…”

“…Another recently released product for cell therapy grade use is recombinant human laminin (Laminin‐521, BioLamina). Specifically, Laminin 521 has been demonstrated to work as a plate coating for culture of both ESC and iPSC lines , as well as for differentiation of stem cells to other cell types .…”

Potential of Induced Pluripotent Stem Cells (iPSCs) for Treating Age-Related Macular Degeneration (AMD)

“…These scaffolds can be made of materials that include collagen and poly(lactic-co-glycolic acid) (PLGA) and it has been demonstrated that a basal support membrane is critical to long-term RPE survival after implantation [56]. It will be of interest to determine the most efficient transplantation method as both single cell suspensions and membrane supports show efficacy [56,57]. …”