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

ABSTRACT: Human 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.

SUBMITTER: Plaza Reyes A

PROVIDER: S-EPMC4720022 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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

Plaza Reyes Alvaro A Petrus-Reurer Sandra S Antonsson Liselotte L Stenfelt Sonya S Bartuma Hammurabi H Panula Sarita S Mader Theresa T Douagi Iyadh I André Helder H Hovatta Outi O Lanner Fredrik F Kvanta Anders A

Stem cell reports 20151224 1

Human 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 ...[more]

PMID: 26724907

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Project description:PURPOSE:Transplantation of human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells offers the potential for benefit in macular degeneration. Previous trials have reported improved visual acuity (VA), but lacked detailed analysis of retinal structure and function in the treated area. DESIGN:Phase 1/2 open-label dose-escalation trial to evaluate safety and potential efficacy (clinicaltrials.gov identifier, NCT01469832). PARTICIPANTS:Twelve participants with advanced Stargardt disease (STGD1), the most common cause of macular degeneration in children and young adults. METHODS:Subretinal transplantation of up to 200?000 hESC-derived RPE cells with systemic immunosuppressive therapy for 13 weeks. MAIN OUTCOME MEASURES:The primary end points were the safety and tolerability of hESC-derived RPE cell administration. We also investigated evidence of the survival of transplanted cells and measured retinal structure and function using microperimetry and spectral-domain OCT. RESULTS:Focal areas of subretinal hyperpigmentation developed in all participants in a dose-dependent manner in the recipient retina and persisted after withdrawal of systemic immunosuppression. We found no evidence of uncontrolled proliferation or inflammatory responses. Borderline improvements in best-corrected VA in 4 participants either were unsustained or were matched by a similar improvement in the untreated contralateral eye. Microperimetry demonstrated no evidence of benefit at 12 months in the 12 participants. In one instance at the highest dose, localized retinal thinning and reduced sensitivity in the area of hyperpigmentation suggested the potential for harm. Participant-reported quality of life using the 25-item National Eye Institute Visual Function Questionnaire indicated no significant change. CONCLUSIONS:Subretinal hyperpigmentation is consistent with the survival of viable transplanted hESC-derived RPE cells, but may reflect released pigment in their absence. The findings demonstrate the value of detailed analysis of spatial correlation of retinal structure and function in determining with appropriate sensitivity the impact of cell transplantation and suggest that intervention in early stage of disease should be approached with caution. Given the slow rate of progressive degeneration at this advanced stage of disease, any protection against further deterioration may be evident only after a more extended period of observation.

Project description:BackgroundHuman embryonic stem cell-derived retinal pigment epithelial cells (hESC-derived RPE) are a promising source for cell-replacement therapy to treat retinal degenerative diseases, but research on RPE cryopreservation is limited. This study aimed to determine the best phase for RPE cryopreservation to preserve the post-thaw function and uncover the mechanism underlying RPE freezing tolerance.MethodshESC-derived RPE cells were cryopreserved at various time points after seeding. After thawing, the survival and attachment rates, RPE marker gene expression, apical-basal polarity, PEDF secretion, transepithelial resistance, and phagocytotic ability of post-thaw RPE cells were evaluated. RNA sequencing was performed on RPE cells at three-time points, differentially expressed genes were identified, and gene ontology, Kyoto encyclopedia of genes and genomes, and protein-protein interaction analyses were used to investigate the key pathways or molecules associated with RPE cell freezing tolerance.ResultsRPE frozen at passage 2 day 5 (P2D5) had the highest cell viability and attachment after thawing. They also retained properly localized expression of RPE marker genes and biological functions such as PEDF secretion, high transepithelial resistance, and phagocytic ability. The RNA-sequencing analysis revealed that RPE cells at P2D5 expressed high levels of cell cycle/DNA replication and ECM binding associated genes, as well as THBS1, which may serve as a possible hub gene involved in freezing tolerance. We also confirmed that the RPE cells at P2D5 were in the exponential stage with active DNA replication.ConclusionsWe propose that freezing hESC-derived RPE cells during their exponential phase results in the best post-thawing outcome in terms of cell viability and preservation of RPE cell properties and functions. The high expression levels of the cell cycle and ECM binding associated genes, particularly THBS1, may contribute to better cell recovery at this stage.

Dataset Information

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

Publications

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

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