Dataset Information

Elucidating the corneal endothelial cell proliferation capacity through an interspecies transcriptome comparison

ABSTRACT: The regenerative capacity of corneal endothelial cells (CECs) differs between species; in bigger mammals such as humans and sheep, CECs are arrested in a non-proliferative state. Damage to these cells can compromise its function leading to corneal opacity and impaired vision. Corneal transplantation is the current treatment for the recovery of a clear eyesight, but the donor tissue demand is higher than the offer and there is the need to develop novel treatment modalities. In contrast, rabbit CECs retain a high proliferative profile and have the capacity to repopulate the endothelium upon injury. There is currently a lack of fundamental knowledge to explain the difference in the proliferation capacity of these cells across species. Gaining information on the transcriptomic differences across species could allow the identification of crucial proliferation drivers of CEC proliferation to develop novel regenerative medicine therapies. In this study we have analyzed at the transcriptomic level corneal endothelial samples originating from human, sheep, and rabbit. To understand the differences across the CECs proliferation capacity in each species, we have generated an automated pipeline for the analysis of pathways with different activity. Our results revealed that 52 pathways had commonly different activity when comparing species with non-proliferative endothelium (human and sheep) to species with proliferative endothelium (rabbit).

ORGANISM(S): Ovis aries Homo sapiens Oryctolagus cuniculus

PROVIDER: GSE222458 | GEO | 2023/01/20

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

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).

Project description:Corneal endothelial cells (CECs) are critical to maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cells (PBMC)-originated induced pluripotent stem cells (iPSCs)-derived CECs. We isolated PBMC and programmed the mononuclear cells to generate iPSCs. Subsequently, the PBMC-originated iPSCs were differentiated to CECs. The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent, and CECs-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSCs-derived CECs and human corneal endothelium (CE) were examined by mass spectrometry-based proteome sequencing. The PBMC-originated iPSCs expressed pluripotent-specific markers at levels similar to expression in H9 human embryonic stem cells (hESCs). Phase contrast microscopy illustrated that iPSCs-derived CECs are tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CECs-associated markers were expressed at many orders of magnitude higher in iPSCs-derived CECs at days 13, 20, and 30 compared to their respective levels in iPSCs. Importantly, only residual expression levels of pluripotency markers were detected in iPSCs-derived CECs. Mass spectrometry-based proteome profiling identified 10,575 proteins in iPSCs-derived CECs. In parallel, we completed proteome profiling of the human CE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSCs-derived CECs suggesting a 90.82% overlap between the iPSCs-derived CECs and human CE proteomes. Importantly, cryopreservation of iPSCs-derived CECs did not affect the tight adherence of CECs, and their hexagonal-like shape while expressing high levels of CECs-associated markers. We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the human CE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSCs-derived CECs.

Project description:Important remodeling of the extracellular matrix is a hallmark of corneal wound healing. Besides the massive secretion of fibronectin (FN) that characterizes the early step of that process, alterations in the secretion of collagens also occurs as part of this wound healingresponse. In this study, we examined whether expression of the gene encoding the M-NM-15 subunit from the FN binding integrin M-NM-15M-NM-21 changes as corneal epithelial cells (CECs) are cultured in the presence of collagens. Responsiveness of the M-NM-15 gene toward collagen was determined by transfection of recombinant plasmids bearing the CAT reporter gene under the control of various segments from the human M-NM-15 promoter into primary cultured rabbit (RCECs) and human (HCECs) CECs cultured on BSA or on collagens. Electrophoretic mobility shift assays (EMSAs) and Western blot analyses were used to monitor both the DNA binding and expression of the transcription factors required to ensure basal transcription of the M-NM-15 gene. The influence collagens on the patterns of genes expressed by HCECs was also studied be gene profiling on microarrays. All collagen types repressed the transcriptional activity directed by the full-length M-NM-15 promoter/CAT construct in confluent CECs. A moderate increase in M-NM-15 promoter activity was observed in subconfluent RCECs grown on CIV but not on CI. These collagen-dependent regulatory influences also correlated with alterations in either the expression or the DNA binding of the transcription factors Sp1/Sp3, NFI and AP-1 that ensure basal transcription of the M-NM-15 gene. Gene profiling on microarray revealed that CI more profoundly alter the pattern of genes expressed by HCECs than what is observed with CIV. Collagens considerably suppressed expression of the M-NM-15 gene in CECs at confluence suggesting that the sustained staining for CI and CIV after corneal injury may play a role in controlling adhesion to the temporary FN matrix and further differentiation of CECs into suprabasal epithelial cells through a mechanism involving the M-NM-15M-NM-21 integrin. Primary cultures of human corneal epithelial cells cultivated on BSA (number of replicates: 2), Collagen type I (number of replicates: 2) and Collagen type IV (number of replicates: 2) matrix.

Dataset Information

Elucidating the corneal endothelial cell proliferation capacity through an interspecies transcriptome comparison

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure