scholarly journals Establishment of Novel Limbus-Derived, Highly Proliferative ABCG2+/ABCB5+ Limbal Epithelial Stem Cell Cultures

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Eung Kweon Kim ◽  
Ga-Hyun Lee ◽  
Boram Lee ◽  
Yong-Sun Maeng
Keyword(s):  
Stem Cell ◽  
Epithelial Cells ◽  
Telomerase Activity ◽  
Stem Cell Markers ◽  
Success Rates ◽  
Epithelial Stem Cells ◽  
Double Positive ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial

Homeostasis and regeneration of corneal epithelia are sustained by limbal epithelial stem cells (LESCs); thus, an LESC deficiency is a major cause of blindness worldwide. Despite the generally promising results of cultivated LESC transplantation, it has been limited by variations in long-term success rates, the use of xenogeneic and undefined culture components, and a scarcity of donor tissues. In this study, we identified the culture conditions required to expand LESCs in vitro and established human limbus-derived highly proliferative ABCG2+/ABCB5+ double-positive LESCs. These LESCs exhibited the LESC marker profile and differentiated into corneal epithelial cells. In addition, cultured LESCs expressed high levels of the stem cell markers Sox2, Oct4, c-Myc, and Klf4, had high telomerase activity, and had stable, normal genomes. These results suggest that our novel cultivation protocol affects the phenotype and differentiation capacity of LESCs. From the limbus, which contains a heterogenous cell population, we have derived highly proliferative ABCG2+/ABCB5+ double-positive cells with the ability to differentiate into corneal epithelial cells. This study opens a new avenue for investigation of the molecular mechanism of LESC maintenance and expansion in vitro and may impact the treatment of corneal disease, particularly corneal blindness due to an LESC deficiency.

scholarly journals Limbal Niche Cells in Three-Dimensional Matrigel Induced Dedifferentiation of Mature Corneal Epithelial Cells toward a Progenitor State

2020 ◽  
Author(s):  
Hui Zhu ◽  
Wei Wang ◽  
Lingjuan Xu ◽  
Menglin Jiang ◽  
Yongyao Tan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Epithelial Cells ◽  
Single Cells ◽  
Three Dimensional ◽  
Stem Cell Marker ◽  
Epithelial Stem Cells ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Epithelial Marker

ABSTRACTPurposeTo investigate the possibility and the key factors of stably committed mature corneal epithelial cells dedifferentiate into corneal epithelial stem cells in vitro.MethodsMature cornea epithelia cell (MCEC) sheets or limbal epithelial progenitor cell (LEPC) sheets were isolated from central corneas or limbal segments by Dispase II and further digested with 0.25% trypsin/1 mM EDTA (T/E) to yield single cells. Limbal niche cells (LNC) were isolated from the limbal stroma by collagenase A and expanded on 5% Matrigel coated plastic. Single MCECs were seeded on 50% Matrigel with or without LNC culturing for 10 days, regarding as three-dimensional MCEC (3D-MCEC) group or three-dimensional MCEC+LNC (3D-MCEC+LNC) group. Expression of CK12, p63α, PCK, Vimentin were analyzed with immunofluorescence staining.ResultsThe expression of mature cornea epithelial marker (CK12) in MCEC was higher than that in LEPC (P=0.020) but epithelial stem cell marker (p63α) was lower than that in LEPC (P=0.000). When seeded in 3D Matrigel, single MCEC cells could form spheres within 72 hours, and the expression of CK12 reduced (P=0.005) and the expression of p63α also reduced to zero (P=0.000) compared to MCEC. Serial passages of LNC which were expanded in coated Matrigel could form spheres in 3D Matrigel. After mixing MCECs with LNC, rounder spheres emerged within 24 hours which consisted of both epithelia cells (PCK+/Vim-) and LNC (PCK-/Vim+). Moreover, epithelia cells in 3D-MCEC+LNC group expressed less CK12 and more p63α than those in MCEC group (P=0.043, 0.000). Besides, the diameter of spheres in 3D-MCEC+LNC group were larger than that in 3D-MCEC group (P=0.000).ConclusionHuman LNC and three-dimensional Matrigel could induce the dedifferentiation of mature corneal epithelial cells into corneal epithelial stem cells.

In Vitro Toxicity of Netilmicin and Ofloxacin on Corneal Epithelial Cells

Cornea ◽  
2003 ◽  
Vol 22 (5) ◽  
pp. 468-472 ◽  
Author(s):  
Anna Claudia Scuderi ◽  
Grazia Maria Paladino ◽  
Clara Marino ◽  
Francesco Trombetta
Keyword(s):  
Epithelial Cells ◽  
In Vitro Toxicity ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial

Recombinant TIMP-1 and -2 enhance the proliferation of rabbit corneal epithelial cells in vitro and the spreading of rabbit corneal epithelium in situ

1998 ◽  
Vol 17 (1) ◽  
pp. 47-52 ◽  
Author(s):  
Shizuya Saika ◽  
Yoshiji Kawashima ◽  
Yuka Okada ◽  
Sai-Ichi Tanaka ◽  
Osamu Yamanaka ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Corneal Epithelium ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial

A 300 bp 5′-upstream sequence of a differentiation-dependent rabbit K3 keratin gene can serve as a keratinocyte-specific promoter

1993 ◽  
Vol 105 (2) ◽  
pp. 303-316 ◽  
Author(s):  
R.L. Wu ◽  
S. Galvin ◽  
S.K. Wu ◽  
C. Xu ◽  
M. Blumenberg ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Transcription Initiation ◽  
Gene Transfection ◽  
Consensus Sequence ◽  
Upstream Sequence ◽  
Basal Cells ◽  
Epithelial Stem Cells ◽  
Keratin Gene ◽  
Corneal Epithelial

Keratinocytes of the suprabasal compartment of many stratified epithelia synthesize as a major differentiation product a keratin pair, consisting of an acidic and a basic keratin, which accounts for 10–20% of the newly synthesized proteins. While genes of several differentiation-related keratins have been cloned and studied, relatively little is known about the molecular basis underlying their tissue-specific and differentiation-dependent expression. We have chosen to study, as a prototype of these genes, the gene of K3 keratin, which has the unique property of being expressed in the majority of corneal epithelial basal cells but suprabasally in peripheral cornea, the site of corneal epithelial stem cells. Using a monoclonal antibody, AE5, specific for K3 keratin, and a fragment of human K3 gene as probes, we have isolated several cDNA and genomic clones of rabbit K3 keratin. One genomic clone has been sequenced and characterized, and the identity of its coding sequence with that of cDNAs indicates that it corresponds to the single, functional rabbit K3 gene. Transfection assays showed that its 3.6 kb 5′-upstream sequence can drive a chloramphenicol acetyl transferase (CAT) reporter gene to express in cultured corneal and esophageal epithelial cells, but not in mesothelial and kidney epithelial cells or fibroblasts, all of rabbit origin. Serial deletion experiments narrowed this keratinocyte-specific promoter to within -300 bp upstream of the transcription initiation site. Its activity is not regulated by the coding or 3′-noncoding sequences that have been tested so far. This 300 bp 5′-upstream sequence of K3 keratin gene, which can function in vitro as a keratinocyte-specific promoter, contains two clusters of partially overlapping motifs, one with an NFkB consensus sequence and another with a GC box. The combinatorial effects of these multiple motifs and their cognate binding proteins may play an important role in regulating the expression of this tissue-restricted and differentiation-dependent keratin gene.

The Effect of Sodium Hyaluronate on the Growth of Rabbit Corneal Epithelial Cells In Vitro

1996 ◽  
Vol 12 (4) ◽  
pp. 409-415 ◽  
Author(s):  
TATSUYA MIYAZAKI ◽  
SATOSHI MIYAUCHI ◽  
TAKEHIKO NAKAMURA ◽  
SAWAKO TAKESHITA ◽  
KATSUYUKI HORIE
Keyword(s):  
Epithelial Cells ◽  
Sodium Hyaluronate ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial
Sign in / Sign up

Export Citation Format

Share Document