Project description:Corneal epithelial cells (CECs) are required for corneal transparency and visual function, and corneal injuries may cause corneal blindness. Skin epidermal stem cells (SESCs), which share the same origin with CECs and have the potential of multi-directional differentiation are ideal seed cells for tissue engineered corneal construction to treat corneal blindness. To identify critical genes and pathways that modulate transdifferentiation from SESCs to CECs, we isolated and cultured the sheep SESCs and CECs and then compared gene expression of SESCs and CECs using microarray.
Project description:To investigate the microRNA expression in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, we have employed Human microRNA Microarray V2 (Agilent) as a screening platform to identify specific microRNAs. We discovered a differential expression of 18 microRNAs against central corneal (CC) epithelia, which contains late transit amplifying cells and terminally differentiated cells. Among them, cluster miR-143/145 was expressed strongly in LPC but at low levels in CC epithelia and this was validated by real-time PCR and locked nucleic acid-based in situ hybridization.
Project description:To investigate the microRNA expression in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, we have employed Human microRNA Microarray V2 (Agilent) as a screening platform to identify specific microRNAs. We discovered a differential expression of 18 microRNAs against central corneal (CC) epithelia, which contains late transit amplifying cells and terminally differentiated cells. Among them, cluster miR-143/145 was expressed strongly in LPC but at low levels in CC epithelia and this was validated by real-time PCR and locked nucleic acid-based in situ hybridization. LPC and CC epithelia, separated by 1-mm in width, were dissected from human cornea for small RNA extraction. Total RNA was extracted by Trizol/chloroform and purified with RNeasy mini spin column. RNA samples with 28S/18S ratios in the range of 1.4 to 1.8 were used for microRNA profiling using an Agilent Human microRNA Microarray V2 platform.
Project description:Integrity of the cornea, the most anterior part of the eye is indispensable for vision. 45 million individuals are bilaterally blind and another 135 millions have severely impaired vision in both eyes because of loss of corneal transparency; treatments range from local medications to corneal transplants and more recently to stem cell therapy. The corneal epithelium is a squamous epithelium that is constantly renewing with a vertical turnover of seven to fourteen days in many mammals3. Identification of slow cycling cells (label-retaining cells or LRCs) in the limbus of the mouse has led to the notion that the limbus is the niche for the stem cells responsible for the long-term renewal of the cornea4; hence, the corneal epithelium is supposedly renewed by cells generated at and migrating from the limbus, in striking opposition to other squamous epithelia in which each resident stem cell has in charge a limited area of epithelium. Here, we show that the corneal epithelium of the mouse can be serially transplanted, is self-maintained and contains oligopotent stem cells with the capacity to generate goblet cells if provided with a conjunctival environment. In addition, the entire ocular surface of the pig, including the cornea, contains oligopotent stem cells (holoclones) with the capacity to generate individual colonies of corneal and conjunctival cells; hence, the limbus is not the only niche for corneal stem cells and corneal renewal is not different from other squamous epithelia. Experiment Overall Design: Expression profile difference between keratinocyte clones from conjunctiva and cornea (3 individual clones in each group)
Project description:Integrity of the cornea, the most anterior part of the eye is indispensable for vision. 45 million individuals are bilaterally blind and another 135 millions have severely impaired vision in both eyes because of loss of corneal transparency; treatments range from local medications to corneal transplants and more recently to stem cell therapy. The corneal epithelium is a squamous epithelium that is constantly renewing with a vertical turnover of seven to fourteen days in many mammals3. Identification of slow cycling cells (label-retaining cells or LRCs) in the limbus of the mouse has led to the notion that the limbus is the niche for the stem cells responsible for the long-term renewal of the cornea4; hence, the corneal epithelium is supposedly renewed by cells generated at and migrating from the limbus, in striking opposition to other squamous epithelia in which each resident stem cell has in charge a limited area of epithelium. Here, we show that the corneal epithelium of the mouse can be serially transplanted, is self-maintained and contains oligopotent stem cells with the capacity to generate goblet cells if provided with a conjunctival environment. In addition, the entire ocular surface of the pig, including the cornea, contains oligopotent stem cells (holoclones) with the capacity to generate individual colonies of corneal and conjunctival cells; hence, the limbus is not the only niche for corneal stem cells and corneal renewal is not different from other squamous epithelia. Keywords: Cell type comparison
Project description: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).
Project description:The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells, while the non-transparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human limbal stem cells from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors that define specific cell fates, and established their regulatory hierarchy. Single-cell RNA-seq analyses of the cornea and the epidermis confirmed these shared and cell type-specific transcription factors. Notably, the shared and limbal stem cell-specific transcription factors can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the limbal stem cell fate and its association with corneal opacity.
Project description:The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells, while the non-transparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human limbal stem cells from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors that define specific cell fates, and established their regulatory hierarchy. Single-cell RNA-seq analyses of the cornea and the epidermis confirmed these shared and cell type-specific transcription factors. Notably, the shared and limbal stem cell-specific transcription factors can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the limbal stem cell fate and its association with corneal opacity.
Project description:The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells, while the non-transparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human limbal stem cells from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors that define specific cell fates, and established their regulatory hierarchy. Single-cell RNA-seq analyses of the cornea and the epidermis confirmed these shared and cell type-specific transcription factors. Notably, the shared and limbal stem cell-specific transcription factors can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the limbal stem cell fate and its association with corneal opacity.