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.

doi:10.1167/iovs.05-0351

Cited by 17 publications

(7 citation statements)

References 38 publications

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“…60,61 Moreover, the survival signaling pathway involving EGFR, and PI3-kinase/Akt is important in LESC expansion, and proliferation without inducing cell differentiation. 62,63 We have also shown here that epithelial wound healing and marker expression in diabetic corneas were further normalized when M10 and CF silencing was combined with c-met overexpression. This Combo treatment accelerated wound healing in diabetic corneas even slightly beyond the normal range, and had an additive effect to both shRNA and c-met treatments alone (Fig.…”

Section: Discussionsupporting

confidence: 56%

Enhanced Wound Healing, Kinase and Stem Cell Marker Expression in Diabetic Organ-Cultured Human Corneas Upon MMP-10 and Cathepsin F Gene Silencing

Saghizadeh

Epifantseva

Hemmati

et al. 2013

Invest. Ophthalmol. Vis. Sci.

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Section: Discussionsupporting

confidence: 56%

Enhanced Wound Healing, Kinase and Stem Cell Marker Expression in Diabetic Organ-Cultured Human Corneas Upon MMP-10 and Cathepsin F Gene Silencing

Saghizadeh

Epifantseva

Hemmati

et al. 2013

Invest. Ophthalmol. Vis. Sci.

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“…The attenuation of the mitogenic activity of PEDF by specific inhibitor of p38 MAPK further confirm the role of p38 MAPK in LSC proliferation induced by PEDF or its 44‐mer peptide. Ex vivo expansion of human LSCs on intact AM is in part supported by ERK‐mediated survival signaling, not p38 MAPK . This difference of signaling mechanism may come from difference of cell culture environment, particularly the involvement of cells and supporting matrix.…”

Section: Discussionmentioning

confidence: 99%

PEDF promotes self-renewal of limbal stem cell and accelerates corneal epithelial wound healing

Chen

et al. 2013

Stem Cells

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Limbal epithelial stem cell (LSC) transplantation is a prevalent therapeutic method for patients with LSC deficiency. The maintenance of stem cell characteristics in the process of culture expansion is critical for the success of ocular surface reconstruction. Pigment epithelialderived factor (PEDF) increased the numbers of holoclone in LSC monolayer culture and preserved the stemness of LSC in suspension culture by evidence of DNp63a, Bmi-1, and ABCG2 expression. BrdU pulse-labeling assay also demonstrated that PEDF stimulated LSCs proliferation. In air-lift culture of limbal equivalent, PEDF was capable of increasing the numbers of DNp63a-positive cells. The mitogenic effect of PEDF was found to be mediated by the phosphorylations of p38 MAPK and STAT3 in LSCs. Synthetic 44-mer PEDF (residues 78-121) was as effective as the full length PEDF in LSC expansion in suspension culture and limbal equivalent formation, as well as the activation of p38 MAPK and STAT3. In mice subjecting to mechanical removal of cornea epithelium, 44-mer PEDF facilitated corneal wound healing. Microscopically, 44-mer PEDF advanced the early proliferative response in limbus, increased the proliferation of DNp63a-positive cells both in limbus and in epithelial healing front, and assisted the repopulation of limbus in the late phase of wound healing. In conclusion, the capability of expanding LSC in cell culture and in animal indicates the potential of PEDF and its fragment (e.g., 44-mer PEDF) in ameliorating limbal stem cell deficiency; and their uses as therapeutics for treating corneal wound.

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“…Key signaling intermediates previously associated with corneal wound healing (Sharma et al 2003; Saika et al 2004; Xu et al 2009; Saghizadeh et al 2010a, 2010b, 2013; Xu and Yu, 2011; 2013), including phosphorylated/activated EGFR, Akt, and p38 MAPK, had higher expression in treated corneas. It should be noted that EGFR-Akt axis is important for expansion of undifferentiated LESC (He et al 2006). Additionally, p38 can influence LESC marker expression including ΔNp63α, and their growth factor-induced self-renewal (Cheng et al 2009; Ho et al 2013).…”

Section: Discussionmentioning

confidence: 99%

Normalization of wound healing and stem cell marker patterns in organ-cultured human diabetic corneas by gene therapy of limbal cells

Saghizadeh

Dib

Brunken

et al. 2014

Experimental Eye Research

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Overexpression of c-met and suppression of matrix metalloproteinase-10 (MMP-10) and cathepsin F genes was previously shown to normalize wound healing, epithelial and stem cell marker patterns in organ-cultured human diabetic corneas. We now examined if gene therapy of limbal cells only would produce similar effects. Eight pairs of organ-cultured autopsy human diabetic corneas were used. One cornea of each pair was treated for 48 hours with adenoviruses (Ad) harboring full-length c-met mRNA or a mixture (combo) of Ad with c-met and shRNA to MMP-10 and cathepsin F genes. Medium was kept at the limbal level to avoid transduction of central corneal epithelium. Fellow corneas received control Ad with EGFP gene. After additional 5 (cmet) or 10 days (combo) incubation, central corneal epithelial debridement with nheptanol was performed, and wound healing times were determined microscopically. Corneal cryostat sections were immunostained for diabetic and putative limbal stem cell markers, α3β1 integrin, nidogen-1, fibronectin, laminin γ3 chain, ΔNp63α, keratins 14, 15, and 17, as well as for activated signaling intermediates, phosphorylated EGFR, Akt, and p38. Limbal c-met overexpression significantly accelerated healing of 8.5-mm epithelial wounds over EGFP controls (6.3 days vs. 9.5 days, p<0.02). Combo treatment produced a similar result (6.75 days vs. 13.5 days, p<0.03). Increased immunostaining vs. EGFP controls for most markers and signaling intermediates accompanied c-met gene or combo transduction. Gene therapy of limbal epithelial stem cell compartment has a beneficial effect on the diabetic corneal wound healing and on diabetic and stem cell marker expression, and shows potential for alleviating symptoms of diabetic keratopathy.

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Signaling-Transduction Pathways Required for Ex Vivo Expansion of Human Limbal Explants on Intact Amniotic Membrane

Abstract: 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.

Cited by 17 publications

References 38 publications

Enhanced Wound Healing, Kinase and Stem Cell Marker Expression in Diabetic Organ-Cultured Human Corneas Upon MMP-10 and Cathepsin F Gene Silencing

Enhanced Wound Healing, Kinase and Stem Cell Marker Expression in Diabetic Organ-Cultured Human Corneas Upon MMP-10 and Cathepsin F Gene Silencing

PEDF promotes self-renewal of limbal stem cell and accelerates corneal epithelial wound healing

Normalization of wound healing and stem cell marker patterns in organ-cultured human diabetic corneas by gene therapy of limbal cells

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