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Greater growth potential of p63-positive epithelial cell clusters maintained in human limbal epithelial sheets.

ABSTRACT: To determine the growth potential of p63-positive cell clusters maintained in human limbal epithelial sheets.Intact human limbal epithelial sheets were isolated from corneoscleral rims and cultured with or without being rendered into single cells by trypsinization on either plastic or 3T3 fibroblast feeder layers. Clonal growth on 3T3 fibroblast feeder layers was compared between monolayers in sheets or single cells and between areas with or without laser-microdissected, p63-enriched cell clusters. Immunostaining, immunoblot analysis, and reverse transcription-polymerase chain reaction of such differentiation markers as keratin (K)-3 and -12 and such progenitor markers as p63, ABCG2, and MDR-1 were also compared.Clusters of small p63-positive cells were enriched in limbal palisades of dispase-isolated epithelial sheets immediately or after brief cultivation on plastic in SHEM. Clonal growth of p63-rich cell clusters was higher than areas without clusters (P < 0.001). Clonal growth of epithelial monolayers derived from sheets was also higher than that derived from single cells (P < 0.01). Although expression of ABCG2 and MDR-1 transcripts was similar, cells from epithelial sheets expressed higher protein levels of p63 and lower protein levels of K3 and -12 than single cells, whether they were cultured on plastic or as 3T3 fibroblast feeder layers.Limbal palisades contain clusters of p63-rich progenitor cells. Maintenance of such cluster architecture during ex vivo expansion yields higher growth potential than being dispersed into single cells.

SUBMITTER: Kawakita T

PROVIDER: S-EPMC2846109 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Greater growth potential of p63-positive epithelial cell clusters maintained in human limbal epithelial sheets.

Kawakita Tetsuya T Shimmura Shigeto S Higa Kazunari K Espana Edgar M EM He Hua H Shimazaki Jun J Tsubota Kazuo K Tseng Scheffer C G SC

Investigative ophthalmology & visual science 20090325 10

<h4>Purpose</h4>To determine the growth potential of p63-positive cell clusters maintained in human limbal epithelial sheets.<h4>Methods</h4>Intact human limbal epithelial sheets were isolated from corneoscleral rims and cultured with or without being rendered into single cells by trypsinization on either plastic or 3T3 fibroblast feeder layers. Clonal growth on 3T3 fibroblast feeder layers was compared between monolayers in sheets or single cells and between areas with or without laser-microdis ...[more]

PMID: 19324845

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Project description:Background:Autologous cultivated oral mucosal epithelial transplantation (COMET) is an important treatment for limbal stem cell deficiency. However, peripheral corneal neovascularization after surgery hinders its application. This study aims to employ a culture system using allogenic limbal niche cells (LNCs) instead of mouse-derived 3T3 cells as a feeder layer that could relieve postoperative neovascularization. Methods:Rat oral mucosal epithelial cells (OMECs) were co-cultured with rat LNCs or 3T3 cells. Cultivated oral mucosal epithelial cells (COMECs) of different culture systems were identified by hematoxylin and eosin staining and immunocytochemistry. The expression levels of the angiogenesis-related factors were analyzed by RT-qPCR and western blotting/ELISA. Angiogenic potential was reconfirmed by cell viability and tube formation assays with human umbilical vein endothelial cells (HUVECs). Results:COMECs were obtained from both culture systems successfully. Immunocytochemistry showed approximately equal percentages of positive staining cells for p63? (p?=?0.9177), ABCG2 (p?=?0.526), Ki67 (p?=?0.0987), and CK3 (p?=?0.4000) in COMECs of different groups. RT-qPCR and western blotting/ELISA showed that COMECs of the LNC group expressed a significantly lower amount of basic fibroblast growth factor (bFGF) (p?=?0.0038 for RT-qPCR, p?=?0.0026 for western blotting) but more pigment epithelium-derived factor (PEDF) (p?=?0.0172 for RT-qPCR, p?=?0.0253 for western blotting) and soluble fms-like tyrosine kinase-1 (sFlt-1) (p?<?0.0001 for RT-qPCR, p?=?0.0064 for ELISA) than the COMECs of the 3T3 group. Furthermore, compared with COMECs of the 3T3 group, COMECs of the LNC group could reduce the viability (p?=?0.0002) and tube formation (p?=?0.0002) of HUVECs. Conclusions:LNCs could substitute 3T3 cells for expanding OMECs in vitro, and the COMECs obtained in this system are less likely to induce postsurgical neovascularization, which provides an alternative option for an ex vivo culture system and promotes the application of COMET.

Project description:The roles of limbal epithelial stem cells (LESCs) are widely recognized, but for these cells to be utilized in basic research and potential clinical applications, researchers must be able to efficiently isolate them and subsequently maintain their stemness in vitro. We aimed to develop a biomimetic environment for LESCs involving cells from their in vivo niche and the principle of flow-induced shear stress, and to subsequently demonstrate the potential of this novel paradigm. LESCs, together with neighboring cells, were isolated from the minced limbal tissues of rabbits. At days 8 and 9 of culture, the cells were exposed to a steady flow or intermittent flow for 2 h per day in a custom-designed bioreactor. The responses of LESCs and epithelial cells were assessed at days 12 and 14. LESCs and epithelial cells responded to both types of flow. Proliferation of LESCs, as assessed using a BrdU assay, was increased to a greater extent under steady flow conditions. Holoclones were found under intermittent flow, indicating that differentiation into transient amplifying cells had occurred. Immunofluorescent staining of Bmi-1 suggested that steady flow has a positive effect on the maintenance of stemness. This finding was confirmed by real-time PCR. Notch-1 and p63 were more sensitive to intermittent flow, but this effect was transient. K3 and K12 expression, indicative of differentiation of LESCs into epithelial cells, was induced by flow and lasted longer under intermittent flow conditions. In summary, culture of LESCs in a bioreactor under a steady flow paradigm, rather than one of intermittent flow, is beneficial for both increasing proliferation and maintaining stemness. Conversely, intermittent flow appears to induce differentiation of LESCs. This novel experimental method introduces micro-mechanical stimuli to traditional culture techniques, and has potential for regulating the proliferation and differentiation of LESCs in vitro, thereby facilitating research in this field.

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Greater growth potential of p63-positive epithelial cell clusters maintained in human limbal epithelial sheets.

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Greater growth potential of p63-positive epithelial cell clusters maintained in human limbal epithelial sheets.

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