Unknown,Transcriptomics,Genomics,Proteomics

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Propagation of Human corneal Endothelial Cells M-bM-^@M-^S A Novel Dual Media Approach

ABSTRACT: 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-

ORGANISM(S): Homo sapiens

SUBMITTER: Jodhbir Mehta

PROVIDER: E-GEOD-50212 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Propagation of Human corneal Endothelial Cells M-bM-^@M-^S A Novel Dual Media Approach

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