PURPOSE: The purpose of this study was to identify phenotypic markers of human limbal stem cells in fetal and adult corneas. METHODS: RNA from microscopically dissected superficial limbal and central fetal (18 weeks) corneas was amplified and used to generate P(32)-labeled, reverse-transcribed antisense RNA that was linearly amplified and hybridized to a focused stem cell cDNA microarray. Differential gene expression of fetal limbus was compared with the expression of central cornea. Microarray differential expression experiments were performed on P63-expressing primary cultured limbal epithelial cells (passage 1; Pa1) and primary cells passaged 5 times (Pa5). Semiquantitative RT-PCR assay and immunohistochemistry were performed on fetal and adult corneas and cultured primary limbal epithelial cells, to confirm the results of the microarray experiments. Slow-cycling (pulsed bromodeoxyuridine label-retaining) limbal epithelium in corneal organ culture was studied for the expression of four selected upregulated limbal genes. RESULTS: Of the 266 genes tested, 33 were differentially overexpressed (more than twofold) in the fetal limbus (compared with central cornea) and primary cultured limbal epithelium compared with primary cells after 5 passages. Cytokeratin 15 (CK15) and cytokeratin 14 (CK14) are expressed in limbal basal epithelium and P-cadherin (CDH3) and Wnt-4 expression was restricted to basal and immediate parabasal limbal epithelium of both the adult and fetal corneas). Bromodeoxyuridine label retaining epithelium in corneal organ culture (slow-cycling cells) expressed the four selected limbal upregulated genes. CONCLUSIONS: For the first time, a focused stem cell pathway microarray analysis has been performed on fetal cornea and cultured limbal explant epithelium. CK15, CK14, CDH3, and Wnt-4 are expressed in the basal limbal epithelial cells.
PURPOSE: The purpose of this study was to identify phenotypic markers of human limbal stem cells in fetal and adult corneas. METHODS: RNA from microscopically dissected superficial limbal and central fetal (18 weeks) corneas was amplified and used to generate P(32)-labeled, reverse-transcribed antisense RNA that was linearly amplified and hybridized to a focused stem cell cDNA microarray. Differential gene expression of fetal limbus was compared with the expression of central cornea. Microarray differential expression experiments were performed on P63-expressing primary cultured limbal epithelial cells (passage 1; Pa1) and primary cells passaged 5 times (Pa5). Semiquantitative RT-PCR assay and immunohistochemistry were performed on fetal and adult corneas and cultured primary limbal epithelial cells, to confirm the results of the microarray experiments. Slow-cycling (pulsed bromodeoxyuridine label-retaining) limbal epithelium in corneal organ culture was studied for the expression of four selected upregulated limbal genes. RESULTS: Of the 266 genes tested, 33 were differentially overexpressed (more than twofold) in the fetal limbus (compared with central cornea) and primary cultured limbal epithelium compared with primary cells after 5 passages. Cytokeratin 15 (CK15) and cytokeratin 14 (CK14) are expressed in limbal basal epithelium and P-cadherin (CDH3) and Wnt-4 expression was restricted to basal and immediate parabasal limbal epithelium of both the adult and fetal corneas). Bromodeoxyuridine label retaining epithelium in corneal organ culture (slow-cycling cells) expressed the four selected limbal upregulated genes. CONCLUSIONS: For the first time, a focused stem cell pathway microarray analysis has been performed on fetal cornea and cultured limbal explant epithelium. CK15, CK14, CDH3, and Wnt-4 are expressed in the basal limbal epithelial cells.
Authors: Jeanie Chui; Minas T Coroneo; Lien T Tat; Roger Crouch; Denis Wakefield; Nick Di Girolamo Journal: Am J Pathol Date: 2011-02 Impact factor: 4.307
Authors: Dhruv Sareen; Mehrnoosh Saghizadeh; Loren Ornelas; Michael A Winkler; Kavita Narwani; Anais Sahabian; Vincent A Funari; Jie Tang; Lindsay Spurka; Vasu Punj; Ezra Maguen; Yaron S Rabinowitz; Clive N Svendsen; Alexander V Ljubimov Journal: Stem Cells Transl Med Date: 2014-07-28 Impact factor: 6.940
Authors: Gen Li; Fan Xu; Jie Zhu; Michal Krawczyk; Ying Zhang; Jin Yuan; Sherrinal Patel; Yujuan Wang; Ying Lin; Ming Zhang; Huimin Cai; Daniel Chen; Meixia Zhang; Guiqun Cao; Emily Yeh; Danni Lin; Qiao Su; Wen-wen Li; George L Sen; Natalie Afshari; Shaochen Chen; Richard L Maas; Xiang-Dong Fu; Kang Zhang; Yizhi Liu; Hong Ouyang Journal: J Biol Chem Date: 2015-06-04 Impact factor: 5.157
Authors: Martin N Nakatsu; Zhenhua Ding; Madelena Y Ng; Thuy T Truong; Fei Yu; Sophie X Deng Journal: Invest Ophthalmol Vis Sci Date: 2011-07-01 Impact factor: 4.799