Comparison of characteristics of cultured limbal cells on denuded amniotic membrane and fresh conjunctival, limbal and corneal tissues

This study aimed to evaluate proposed molecular markers related to eye limbal stem cells (SC) and to identify novel associated genes. The expression of a set of genes potentially involved in stemness was assessed in freshly prepared limbal, corneal and conjunctival tissues. PAX6, AC133, K12 and OCT4 were detected in all the tissues and p63+/K3−/K12+/Nodal+/Cx43+ were expressed in conjunctival, p63−/K3+/K12+/Nodal−/Cx43+ in corneal, and p63+/K3−/K12−/Nodal−/Cx43− in limbal tissues. Limbal explants were cultured on human amniotic membrane for 21 days. The cells expressed p63 but not K3, K12, Nodal and Cx43, however, the expression of K3, K12 and Cx43 was detected, and p63 and the high BrdU‐labeling index decreased with more culture. Ultrastructure analysis of the cultured cells showed typically immature organization of intracellular organelles and architecture. Our data suggest that limbal, corneal and conjunctival tissues are heterogeneous with some progenitors. Also, the expression of traditional SC markers may not be a reliable indicator of limbal SC and there is an increasing need to determine factor(s) involved in their stemness.

[1]  J. Piatigorsky,et al.  Serial analysis of gene expression (SAGE) in the rat limbal and central corneal epithelium. , 2006, Investigative ophthalmology & visual science.

[2]  S. Kinoshita,et al.  Clusters of corneal epithelial cells reside ectopically in human conjunctival epithelium. , 2006, Investigative ophthalmology & visual science.

[3]  A. Kicic,et al.  Limbal stem cells: the search for a marker , 2006, Clinical & experimental ophthalmology.

[4]  U. Schlötzer-Schrehardt,et al.  Identification and characterization of limbal stem cells. , 2005, Experimental eye research.

[5]  L. Olson,et al.  Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis. , 2004, Carcinogenesis.

[6]  D. Tan,et al.  Ocular surface stem cells and disease: current concepts and clinical applications. , 2004, Annals of the Academy of Medicine, Singapore.

[7]  D. Harkin,et al.  Analysis of p63 and cytokeratin expression in a cultivated limbal autograft used in the treatment of limbal stem cell deficiency , 2004, British Journal of Ophthalmology.

[8]  De-Quan Li,et al.  Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures. , 2004, Experimental eye research.

[9]  H. Baharvand,et al.  Establishment and in vitro differentiation of a new embryonic stem cell line from human blastocyst. , 2004, Differentiation; research in biological diversity.

[10]  De-Quan Li,et al.  Characterization of Putative Stem Cell Phenotype in Human Limbal Epithelia , 2004, Stem cells.

[11]  John D West,et al.  Corneal development, limbal stem cell function, and corneal epithelial cell migration in the Pax6(+/-) mouse. , 2004, Investigative ophthalmology & visual science.

[12]  S. Tseng,et al.  Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle. , 2004, Experimental eye research.

[13]  H. Dua,et al.  Stem cell differentiation and the effects of deficiency , 2003, Eye.

[14]  S. Tseng,et al.  Modulation of keratin and connexin expression in limbal epithelium expanded on denuded amniotic membrane with and without a 3T3 fibroblast feeder layer. , 2003, Investigative ophthalmology & visual science.

[15]  J. Piatigorsky,et al.  Requirement for Pax6 in corneal morphogenesis: a role in adhesion , 2003, Journal of Cell Science.

[16]  W. Thoreson,et al.  Adult corneal limbal epithelium: a model for studying neural potential of non-neural stem cells/progenitors. , 2002, Developmental biology.

[17]  D. Melton,et al.  "Stemness": Transcriptional Profiling of Embryonic and Adult Stem Cells , 2002, Science.

[18]  S. Tseng,et al.  Phenotypic study of a case with successful transplantation of ex vivo expanded human limbal epithelium for unilateral total limbal stem cell deficiency. , 2002, Ophthalmology.

[19]  S. Tseng,et al.  Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures , 2002, The British journal of ophthalmology.

[20]  N. Koizumi,et al.  Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. , 2001, Ophthalmology.

[21]  Kousaku Okubo,et al.  Characteristics of the Human Ocular Surface Epithelium , 2001, Progress in Retinal and Eye Research.

[22]  D. Harkin,et al.  Phenotypic analyses of limbal epithelial cell cultures derived from donor corneoscleral rims , 2001, Clinical & experimental ophthalmology.

[23]  D. Ponzin,et al.  p63 identifies keratinocyte stem cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[24]  I. Weissman,et al.  Direct isolation of human central nervous system stem cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[25]  S. Kinoshita,et al.  Pathological keratinization of ocular surface epithelium. , 2000, Advances in experimental medicine and biology.

[26]  R. Tsai,et al.  Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. , 2000, The New England journal of medicine.

[27]  K. Pantel,et al.  In vitro differentiation of endothelial cells from AC133-positive progenitor cells , 2000 .

[28]  J. Miyazaki,et al.  Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells , 2000, Nature Genetics.

[29]  H. Dua,et al.  Autologous limbal transplantation in patients with unilateral corneal stem cell deficiency , 2000, The British journal of ophthalmology.

[30]  H. Dua,et al.  Limbal stem cells of the corneal epithelium. , 2000, Survey of ophthalmology.

[31]  W. Huttner,et al.  The Human AC133 Hematopoietic Stem Cell Antigen Is also Expressed in Epithelial Cells and Targeted to Plasma Membrane Protrusions* , 2000, The Journal of Biological Chemistry.

[32]  K. Pantel,et al.  In vitro differentiation of endothelial cells from AC133-positive progenitor cells. , 1999, Blood.

[33]  Srinivas K. Rao,et al.  Limbal allografting from related live donors for corneal surface reconstruction. , 1999, Ophthalmology.

[34]  A. Yang,et al.  p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. , 1998, Molecular cell.

[35]  K. Chew,et al.  The functional relationship between in vivo bromodeoxyuridine labeling index and Ki-67 proliferation index in human breast cancer , 1998, Breast Cancer Research and Treatment.

[36]  T. Sun,et al.  Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation. , 1998, Journal of cell science.

[37]  M. Matić,et al.  Stem cells of the corneal epithelium lack connexins and metabolite transfer capacity. , 1997, Differentiation; research in biological diversity.

[38]  K. Tsubota,et al.  Reconstruction of the corneal epithelium by limbal allograft transplantation for severe ocular surface disorders. , 1995, Ophthalmology.

[39]  S. Tseng,et al.  Limbal autograft transplantation for ocular surface disorders. , 1989, Ophthalmology.

[40]  A. Schermer,et al.  Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells , 1986, The Journal of cell biology.

[41]  D. Ponzin,et al.  Further evaluation of amniotic membrane banking for transplantation in ocular surface diseases , 2004, Cell and Tissue Banking.

[42]  S. Tseng,et al.  Connexin 43 expression and proliferation of human limbal epithelium on intact and denuded amniotic membrane. , 2002, Investigative ophthalmology & visual science.

[43]  S. Tseng,et al.  Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane for treating corneal diseases with total limbal stem cell deficiency. , 2002, Advances in experimental medicine and biology.

[44]  S. Tseng.  Concept and application of limbal stem cells , 1989, Eye.