Abstract
PURPOSE. Ex vivo expansion of limbal epithelial progenitor cells on amniotic membrane (AM) without 3T3 fibroblasts is a new surgical approach to treat limbal stem cell deficiency. Such expansion requires NGF-TrkA-mediated signaling, and this study was conducted to delineate the downstream signaling pathways. METHODS. The human corneolimbal ring was cut into explants and cultured on intact human AM. At day 0 or 10, low-molecular-weight inhibitors were added, whereas the control group received dimethyl sulfoxide (DMSO). The epithelial outgrowth rate was monitored for 17 days, and the epithelial cells were collected for Western blot analysis. RESULTS. In the control, most expansion of human limbal epithelial cells started from the limbus from days 5 to 7 and reached ∼80% confluence at day 17. Compared with the control, the outgrowth was completely inhibited by 50 μM LY294002 or 50 μM SR13668 and was significantly suppressed by 10 μM U0126, but was not affected by 10 μM of either SB203580 or JNK inhibitor 1. The inhibition of outgrowth by LY294002, SR13668, and U0126 was reversible. Western blot analysis showed that phosphorylation of Akt and FKHRL1was abolished by LY294002 and SR13668, but downregulated by U0126, which also abolished phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). The phosphorylation of p38 and JNK MAPK were downregulated or abolished during ex vivo expansion. CONCLUSIONS. Ex vivo expansion of human limbal epithelial progenitor cells on intact AM is mediated by the survival signaling pathway mediated by PI3K-Akt-FKHRL1 and by the mitogenic MAPK pathway mediated by p44/42 at the expense of p38 and JNK MAPK.
| Original language | English |
|---|---|
| Pages (from-to) | 151-157 |
| Number of pages | 7 |
| Journal | Investigative Ophthalmology and Visual Science |
| Volume | 47 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2006 Jan |
| Externally published | Yes |
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ASJC Scopus subject areas
- Ophthalmology
Cite this
Signaling-transduction pathways required for ex vivo expansion of human limbal explants on intact amniotic membrane. / He, Hua; Cho, Hee Tae; Li, Wei; Kawakita, Tetsuya; Jong, Ling; Tseng, Scheffer C G.
In: Investigative Ophthalmology and Visual Science, Vol. 47, No. 1, 01.2006, p. 151-157.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Signaling-transduction pathways required for ex vivo expansion of human limbal explants on intact amniotic membrane
AU - He, Hua
AU - Cho, Hee Tae
AU - Li, Wei
AU - Kawakita, Tetsuya
AU - Jong, Ling
AU - Tseng, Scheffer C G
PY - 2006/1
Y1 - 2006/1
N2 - PURPOSE. Ex vivo expansion of limbal epithelial progenitor cells on amniotic membrane (AM) without 3T3 fibroblasts is a new surgical approach to treat limbal stem cell deficiency. Such expansion requires NGF-TrkA-mediated signaling, and this study was conducted to delineate the downstream signaling pathways. METHODS. The human corneolimbal ring was cut into explants and cultured on intact human AM. At day 0 or 10, low-molecular-weight inhibitors were added, whereas the control group received dimethyl sulfoxide (DMSO). The epithelial outgrowth rate was monitored for 17 days, and the epithelial cells were collected for Western blot analysis. RESULTS. In the control, most expansion of human limbal epithelial cells started from the limbus from days 5 to 7 and reached ∼80% confluence at day 17. Compared with the control, the outgrowth was completely inhibited by 50 μM LY294002 or 50 μM SR13668 and was significantly suppressed by 10 μM U0126, but was not affected by 10 μM of either SB203580 or JNK inhibitor 1. The inhibition of outgrowth by LY294002, SR13668, and U0126 was reversible. Western blot analysis showed that phosphorylation of Akt and FKHRL1was abolished by LY294002 and SR13668, but downregulated by U0126, which also abolished phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). The phosphorylation of p38 and JNK MAPK were downregulated or abolished during ex vivo expansion. CONCLUSIONS. Ex vivo expansion of human limbal epithelial progenitor cells on intact AM is mediated by the survival signaling pathway mediated by PI3K-Akt-FKHRL1 and by the mitogenic MAPK pathway mediated by p44/42 at the expense of p38 and JNK MAPK.
AB - PURPOSE. Ex vivo expansion of limbal epithelial progenitor cells on amniotic membrane (AM) without 3T3 fibroblasts is a new surgical approach to treat limbal stem cell deficiency. Such expansion requires NGF-TrkA-mediated signaling, and this study was conducted to delineate the downstream signaling pathways. METHODS. The human corneolimbal ring was cut into explants and cultured on intact human AM. At day 0 or 10, low-molecular-weight inhibitors were added, whereas the control group received dimethyl sulfoxide (DMSO). The epithelial outgrowth rate was monitored for 17 days, and the epithelial cells were collected for Western blot analysis. RESULTS. In the control, most expansion of human limbal epithelial cells started from the limbus from days 5 to 7 and reached ∼80% confluence at day 17. Compared with the control, the outgrowth was completely inhibited by 50 μM LY294002 or 50 μM SR13668 and was significantly suppressed by 10 μM U0126, but was not affected by 10 μM of either SB203580 or JNK inhibitor 1. The inhibition of outgrowth by LY294002, SR13668, and U0126 was reversible. Western blot analysis showed that phosphorylation of Akt and FKHRL1was abolished by LY294002 and SR13668, but downregulated by U0126, which also abolished phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). The phosphorylation of p38 and JNK MAPK were downregulated or abolished during ex vivo expansion. CONCLUSIONS. Ex vivo expansion of human limbal epithelial progenitor cells on intact AM is mediated by the survival signaling pathway mediated by PI3K-Akt-FKHRL1 and by the mitogenic MAPK pathway mediated by p44/42 at the expense of p38 and JNK MAPK.
UR - http://www.scopus.com/inward/record.url?scp=33644841408&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33644841408&partnerID=8YFLogxK
U2 - 10.1167/iovs.05-0351
DO - 10.1167/iovs.05-0351
M3 - Article
C2 - 16384957
AN - SCOPUS:33644841408
VL - 47
SP - 151
EP - 157
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
SN - 0146-0404
IS - 1
ER -