Ying-Ting Chen

Niche regulation of corneal epithelial stem cells at the limbus

Among all adult somatic stem cells, those of the corneal epithelium are unique in their exclusive location in a defined limbal structure termed Palisades of Vogt. As a result, surgical engraftment of limbal epithelial stem cells with or without ex vivo expansion has long been practiced to restore sights in patients inflicted with limbal stem cell deficiency. Nevertheless, compared to other stem cell examples, relatively little is known about the limbal niche, which is believed to play a pivotal role in regulating self-renewal and fate decision of limbal epithelial stem cells. This review summarizes relevant literature and formulates several key questions to guide future research into better understanding of the pathogenesis of limbal stem cell deficiency and further improvement of the tissue engineering of the corneal epithelium by focusing on the limbal niche.

Suppression of Activation and Induction of Apoptosis in RAW264.7 Cells by Amniotic Membrane Extract

PURPOSEnMacrophages play a pivotal role in initiating, maintaining, and resolving host inflammatory/immune responses but may cause recalcitrant inflammation and tissue damage if not controlled. Clinically, amniotic membrane (AM) transplantation suppresses inflammation in ocular surface reconstruction. Experimentally, the authors and others have reported that AM facilitates macrophage apoptosis. However, it remains unclear whether such anti-inflammatory activity is retained in AM extract (AME).nnnMETHODSnHerein the authors demonstrate in resting and activated (by interferon [IFN]-gamma, lipopolysaccharide [LPS], or IFN-gamma/LPS) murine monocyte/macrophage RAW264.7 cells that AME suppresses cell spreading and reduces actin filaments determined by phalloidin staining and Western blotting of Triton X-100 extracted cell lysate.nnnRESULTSnWestern blot and immunocytochemistry staining showed AME downregulates the expression of such cell surface markers as CD80, CD86, and major histocompatibility complex class 2 antigen. Cell growth/viability is inhibited whereas cell apoptosis is enhanced by AME. Accordingly, secreted proinflammatory cytokines such as TNF-alpha and IL-6 are reduced, but anti-inflammatory cytokine IL-10 is upregulated.nnnCONCLUSIONSnCollectively, these results suggest that, similar to amniotic membrane, AME retains anti-inflammatory activities and does so by downregulating activation and inducing apoptosis in macrophages.

Reversal of myofibroblasts by amniotic membrane stromal extract

Myofibroblasts play an important role in morphogenesis, inflammation, and fibrosis in most tissues. The amniotic membrane stroma can maintain keratocytes in cultures and prevent them from differentiating into myofibroblasts. However, it is unknown whether the AM stroma can also reverse differentiated myofibroblasts. In this study, we found that amniotic membrane stromal cells (AMSCs), which adopted fibroblastic phenotype in vivo, quickly and completely differentiated into myofibroblasts during ex vivo culture in DMEM/FBS on plastic within 2 passages. When cultured on type I collagen, the myofibroblasts maintained their phenotype, however, when these myofibroblasts were re‐seeded onto a cryopreserved amniotic membrane stromal surface, they reversed to the fibroblast phenotype. Moreover, we found that the amniotic membrane stromal extract not only helps maintain primary AMSCs fibroblastic phenotype in vitro, but also can reverse differentiated myofibroblasts back to fibroblasts. This reversal was not coupled with cell proliferation. We concluded that the amniotic membrane stroma contains soluble factors that can regulate the mesenchymal cell differentiation. Further investigation into the identity of these factors and the control mechanisms may unravel a new scar‐reversing strategy. J. Cell. Physiol. 215: 657–664, 2008.

Air Exposure–Induced Squamous Metaplasia of Human Limbal Epithelium

PURPOSEnSquamous metaplasia is a pathologic process that frequently occurs in nonkeratinized stratified ocular surface epithelia. The mechanism for this occurrence is largely unknown except for vitamin A deficiency.nnnMETHODSnHuman limbal explants were cultured under airlift with or without p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 or in a submerged manner for different durations up to 2 weeks. Epithelial cell proliferation, differentiation, limbal stem cell maintenance, and expansion were studied using certain markers such as Ki67, p63, K10 and K12 keratins, filaggrin, Pax6, ABCG-2, and Musashi-1. Expression of phospho-p38 MAPK and its downstream transcription factors, C/EBPalpha and C/EBPbeta, were studied by immunohistochemistry. Epithelial cells harvested from explants after 2 weeks of culturing under different conditions were seeded onto 3T3 feeder layers and cultured for 12 days. The differentiation of clonal epithelial cells was investigated by double staining to K12 and K10 keratins.nnnRESULTSnThe squamous metaplasia model was successfully created by culturing human limbal explants at an air-liquid interface (airlift) for 2 weeks. Increased stratification and hyperproliferation only happened in the limbal, but not the corneal, epithelium in airlift, but not submerged, cultures. Epithelial proliferation was associated with a transient increase of limbal epithelial stem cells. Abnormal epidermal differentiation-evidenced by positive expression of K10 keratin in suprabasal cells and filaggrin in superficial cells-ensued. Clones generated from epithelial cells harvested from airlift culture only expressed K12 keratin without K10. As early as 2 days in airlift cultures, p38 expression emerged in limbal basal epithelial cells and gradually extended to the cytoplasm and nuclei. Furthermore, addition of the p38 inhibitor SB203580 abolished abnormal epidermal differentiation without affecting limbal epithelial proliferation. Expression of C/EBPalpha and C/EBPbeta, downstream of the p38 MAPK signaling pathway, was strongly induced by airlift culture and partially was inhibited by SB203580.nnnCONCLUSIONSnDryness resulting from exposure activates p38 MAPK signaling coupled with abnormal epidermal differentiation without intrinsic alteration of stem cells in the limbus. On the ocular surface, p38 inhibitors may have the potential to revert the pathologic process of squamous metaplasia induced by dryness.

Heterogeneity of limbal basal epithelial progenitor cells.

Although corneal epithelial stem cells (SCs) are located at the limbus between the cornea and the conjunctiva, not all limbal basal epithelial cells are SCs. Using 2 dispase digestions to remove different amounts of limbal basal epithelial cells for cross-sections, flat mounts, and cytospin preparations, double immunostaining to pancytokeratins (PCK) and vimentin (Vim) identified 3 p63+ epithelial progenitors such as PCK−/Vim−, PCK+/Vim+, and PCK+/Vim− and 1 p63− mesenchymal cell, PCK−/Vim+. PCK−/Vim− progenitors had the smallest cell size were 10-20 times more enriched on collagen I-coated dishes in the 5-minute rapid adherent fraction that contained the highest percentage of p63+ cells but the lowest percentage of cytokeratin12+ cells, and gave rise to high Ki67 labeling and vivid clonal growth. In contrast, PCK+/Vim+ and PCK+/Vim− progenitors were found more in the slow-adherent fraction and yielded poor clonal growth. PCK−/Vim− progenitors and clusters of PCK−/Vim+ mesenchymal cells, which were neither melanocytes nor Langerhans cells, were located in the limbal basal region. Therefore, differential expression of PCK and Vim helps identify small PCK−/Vim− cells as the most likely candidate for SCs among a hierarchy of heterogeneous limbal basal progenitors, and their close association with PCK−/Vim+ presumed “niche” cells.