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A SINGLE-CELL RNA-SEQ ANALYSIS UNRAVELS THE HETEROGENEITY OF PRIMARY CULTURED HUMAN CORNEAL ENDOTHELIAL CELLS

ABSTRACT: The corneas is a transparent and avascular tissue located in front of the eye. Its inner surface is lined by a monolayer of corneal endothelial cells (CECs), which maintain the cornea transparent. CECs remain arrested at a non-proliferative state and damage to these cells can compromise their function leading to corneal opacity. The primary culture of donor-derived CECs is a promising cell therapy. It confers the potential to treat multiple patients from a single donor, alleviating the global donor shortage. Nevertheless, this approach has limitations preventing its adoption, particularly culture protocols allow limited expansion of CECs and there is a lack of clear parameters to identify therapy-grade CECs. To address this limitation, a better understanding of the molecular changes arising from the primary culture of CECs is required. Using single-cell RNA sequencing on primary cultured CECs, we identify their variable transcriptomic fingerprint at the single cell level, provide a pseudo temporal reconstruction of the changes arising from primary culture, and suggest markers to assess the quality of primary CEC cultures. This research depicts a deep transcriptomic understanding of the cellular heterogeneity arising from the primary expansion of CECs and sets the basis for further improvement of culture protocols and therapies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE227942 | GEO | 2023/06/06

REPOSITORIES: GEO

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Project description:Human corneal endothelial cells (CECs) are not able to proliferate or regenerate within the eye. However, limited in vitro expansion of primary human CECs has been demonstrated by several laboratories, including ours. Due to the nature of these primary cells, various culture conditions supporting the active proliferation of these cells also drives them towards an endothelial-to-mesenchymal transformation (EMT) into fibroblastic-like cells. The occurrence of such a phenomenon has been observed as early as after the first round of passage. However, it should be noted that the degree of such transformation is highly heterogeneous, due likely to donor variability. In the current study, propagation of primary human CECs was carried out using a novel dual media approach, where M4 (F99: HamM-bM-^@M-^Ys F12/M199, 5% FBS, 20 M-NM-<g/ml ascorbic acid, 1% ITS, 1% antibiotic/antimycotic and 10 ng/ml bFGF) was used to promote the proliferation of the CECs, and M5 (Endo: Human Endothelial-SFM, 5% FBS and 1% antibiotic/antimycotic), was used to stabilized these primary CECs when they reached 80% to 90% confluence.Primary cultures exposed to M5 were able to maintain the unique cellular structure of the human corneal endothelial cells and do not undergo EMT. In order to better understand the observed morphological changes at the molecular level following the switch from the proliferative M4 medium to the maintainance M5 medium, high throughput microarray analysis was performed on human CECs isolated from two donors and cultivated to the third passage. Subsequently, the global gene expression profile of the confluent human CECs expanded in M4 for the first (D17p) and second donor (D18p), were compared to the same set of the confluent human CECs maintained in M5 medium (D17m and D18m) for a further seven days. Primary human CECs were expanded using the dual media approach to the third passage. Total RNA obtained from confluent P3 human CECs samples grown in the proliferative M4 medium (D17p and D18p) were compared to the same set of primary cultures that were exposed to M5 for a further 7 days (D17m and D18m).

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A SINGLE-CELL RNA-SEQ ANALYSIS UNRAVELS THE HETEROGENEITY OF PRIMARY CULTURED HUMAN CORNEAL ENDOTHELIAL CELLS

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