Dataset Information

Wound healing of human embryonic stem cell-derived retinal pigment epithelial cells is affected by maturation stage.

ABSTRACT:

Background

Wound healing of retinal pigment epithelium (RPE) is a complex process that may take place in common age-related macular degeneration eye disease. The purpose of this study was to evaluate whether wounding and wound healing has an effect on Ca²⁺ dynamics in human embryonic stem cell (hESC)-RPEs cultured different periods of time.

Methods

The 9-day-cultured or 28-day-cultured hESC-RPEs from two different cell lines were wounded and the dynamics of spontaneous and mechanically induced intracellular Ca²⁺ activity was measured with live-cell Ca²⁺ imaging either immediately or 7 days after wounding. The healing time and speed were analyzed with time-lapse bright field microscopy. The Ca²⁺ activity and healing speed were analysed with image analysis. In addition the extracellular matrix deposition was assessed with confocal microscopy.

Results

The Ca²⁺ dynamics in hESC-RPE monolayers differed depending on the culture time: 9-day-cultured cells had higher number of cells with spontaneous Ca²⁺ activity close to freshly wounded edge compared to control areas, whereas in 28-day-cultured cells there was no difference in wounded and control areas. The 28-day-cultured, wounded and 7-day-healed hESC-RPEs produced wide-spreading intercellular Ca²⁺ waves upon mechanical stimulation, while in controls propagation was restricted. Most importantly, both wave spreading and spontaneous Ca²⁺ activity of cells within the healed area, as well as the cell morphology of 28-day-cultured, wounded and thereafter 7-day-healed areas resembled the 9-day-cultured hESC-RPEs.

Conclusions

This acquired knowledge about Ca²⁺ dynamics of wounded hESC-RPE monolayers is important for understanding the dynamics of RPE wound healing, and could offer a reliable functionality test for RPE cells. The data presented in here suggests that assessment of Ca²⁺ dynamics analysed with image analysis could be used as a reliable non-invasive functionality test for RPE cells.

SUBMITTER: Abu Khamidakh AE

PROVIDER: S-EPMC6069779 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Publications

Wound healing of human embryonic stem cell-derived retinal pigment epithelial cells is affected by maturation stage.

Abu Khamidakh Amna E AE Rodriguez-Martinez Alejandra A Kaarniranta Kai K Kallioniemi Anne A Skottman Heli H Hyttinen Jari J Juuti-Uusitalo Kati K

Biomedical engineering online 20180731 1

<h4>Background</h4>Wound healing of retinal pigment epithelium (RPE) is a complex process that may take place in common age-related macular degeneration eye disease. The purpose of this study was to evaluate whether wounding and wound healing has an effect on Ca<sup>2+</sup> dynamics in human embryonic stem cell (hESC)-RPEs cultured different periods of time.<h4>Methods</h4>The 9-day-cultured or 28-day-cultured hESC-RPEs from two different cell lines were wounded and the dynamics of spontaneous ...[more]

PMID: 30064430

Similar Datasets

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.

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.

Dataset Information

Wound healing of human embryonic stem cell-derived retinal pigment epithelial cells is affected by maturation stage.

Background

Methods

Results

Conclusions

Publications

Wound healing of human embryonic stem cell-derived retinal pigment epithelial cells is affected by maturation stage.

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