A rabbit anterior cornea replacement derived from acellular porcine cornea matrix, epithelial cells and keratocytes

Pang, Kunpeng; Du, Liqun; Wu, Xinyi

doi:10.1016/j.biomaterials.2010.05.066

Cited by 105 publications

(111 citation statements)

References 36 publications

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“…All have previously been utilized to demonstrate the activated repair subtypes of corneal stromal cells. 8,20,22 All of these markers were positively detected in the 2D-activated stromal cells (Fig. 4O, T and Y) before 3D hydrogel encapsulation.…”

Section: Resultsmentioning

confidence: 95%

See 1 more Smart Citation

Corneal Stromal Cell Plasticity: In Vitro Regulation of Cell Phenotype Through Cell-Cell Interactions in a Three-Dimensional Model

Wilson

Yang²,

Haj³

2014

Tissue Engineering Part A

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In vivo, epithelial cells are connected both anatomically and functionally with stromal keratocytes. Co-culturing aims at recapturing this cellular anatomy and functionality by bringing together two or more cell types within the same culture environment. Corneal stromal cells were activated to their injury phenotype (fibroblasts) and expanded before being encapsulated in type I collagen hydrogels constructs. Three different epithelial-stromal co-culture methods were then examined: epithelial explant; transwell; and the use of conditioned media. The aim was to determine whether the native, inactivated keratocyte cell phenotype could be restored in vitro. Media supplementation with transforming growth factor beta-1 (TGF-b1) was then used to determine whether the inactivated stromal cells retained their plasticity in vitro and could be re-activated to the fibroblast phenotype. Finally, media supplementation with wortmannin was used to inhibit epithelial-stromal cell interactions. Two different nondestructive techniques, spherical indentation and optical coherence tomography, were used to reveal how epithelial-stromal co-culturing with TGF-b1, and wortmannin media supplementation, respectively, affect stromal cell behavior and differentiation in terms of construct contraction and elastic modulus measurement. Cell viability, phenotype, morphology, and protein expression were investigated to corroborate our mechanical findings. It was shown that activated stromal cells could be inactivated to a keratocyte phenotype via co-culturing and that they retained their plasticity in vitro. Activated corneal stromal cells that were fibroblastic in phenotype were successfully reverted to a nonactivated keratocyte cell lineage in terms of behavior and biological properties; and then back again via TGF-b1 media supplementation. It was then revealed that epithelial-stromal interactions can be blocked via the use of wortmannin inhibition. A greater understanding of stromal-epithelial interactions and what mediates them offers great pharmacological potential in the regulation of corneal wound healing, with the potential to treat corneal diseases and injury by which such interactions are vital.

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Section: Resultsmentioning

confidence: 95%

“…All epithelial cultures were immunohistochemically characterized using primary antibodies CK3 and vimentin after 14 days of culture. CK3 is a specific corneal epithelial marker [19][20][21] ; and vimentin has been shown to be expressed in injured epithelial cells. 6 All samples stained positive for CK3 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Corneal Stromal Cell Plasticity: In Vitro Regulation of Cell Phenotype Through Cell-Cell Interactions in a Three-Dimensional Model

Wilson

Yang²,

Haj³

2014

Tissue Engineering Part A

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show abstract

“…Several recent studies have considered the importance of the stromal cells in corneal epithelial tissue engineering and have developed experimental approaches that incorporate stromal cells in the tissue construct. [37][38][39] Our study is the first to use a decellularized human cadaver cornea instead of a xenograft cornea. Decellularized human corneas, while eliminating the theoretical risk of zoonotic infections, are also less likely to induce immunologic responses in a human host that may potentially be seen with xenogenic grafts.…”

Section: Discussionmentioning

confidence: 99%

Decellularized Human Cornea for Reconstructing the Corneal Epithelium and Anterior Stroma

Shafiq

Gemeinhart

Yue

et al. 2012

Tissue Engineering Part C: Methods

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“…Specific primers for the GAPDH gene were used as controls. Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) was run in duplicate using the Light Cycler (Roche) at 95°C for 30 Preparation of posterior acellular porcine corneal matrix lamellae and construction of CEC-like cell sheets Decellularization of porcine corneas was implemented according to the previous protocols in our lab [19,20]. A 1.0-mm-thick, 8.0-mm-diameter APCM lamella containing posterior stromal collagen and Descemet's membrane [ie, posterior acellular porcine corneal matrix (PAPCM) lamella] was prepared using a scalpel under a dissecting microscope.…”

Section: Real-time Reverse Transcription Polymerase Chain Reactionmentioning

confidence: 99%

Isolation and Transplantation of Corneal Endothelial Cell–Like Cells Derived from In-Vitro-Differentiated Human Embryonic Stem Cells

Zhang

Pang

2014

Stem Cells and Development

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The maintenance of corneal dehydration and transparency depends on barrier and pump functions of corneal endothelial cells (CECs). The human CECs have no proliferation capacity in vivo and the ability to divide in vitro under culture conditions is dramatically limited. Thus, the acquisition of massive cells analogous to normal human CECs is extremely necessary whether from the perspective of cellular basic research or from clinical applications. Here we report the derivation of CEC-like cells from human embryonic stem cells (hESCs) through the periocular mesenchymal precursor (POMP) phase. Using the transwell coculture system of hESCs with differentiated human corneal stromal cells, we induced hESCs to differentiate into POMPs. Then, CEC-like cells were derived from POMPs with lens epithelial cell-conditioned medium. Within 1 week, CEClike cells that expressed the corneal endothelium (CE) differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2 were detectable. Fluorescence-activated cell sorting (FACS)-based isolation of the Ncadherin/vimentin dual-positive population enriches for CEC-like cells. The isolated CEC-like cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) and seeded onto posterior acellular porcine corneal matrix lamellae to construct the CEC-like cell sheets. Pump function parameters of the CEClike cell sheets approximated those of human donor corneas. Importantly, when the CEC-like cell sheets were transplanted into the eyes of rabbit CE dysfunction models, the corneal transparency was restored gradually. In conclusion, CEC-like cells derived from hESCs displayed characteristics of native human CECs. This renewable source of human CECs offers massive cells for further studies of human CEC biological characteristics and potential applications of replacement therapies as substitution for donor CECs in the future.

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A rabbit anterior cornea replacement derived from acellular porcine cornea matrix, epithelial cells and keratocytes

Cited by 105 publications

References 36 publications

Corneal Stromal Cell Plasticity: In Vitro Regulation of Cell Phenotype Through Cell-Cell Interactions in a Three-Dimensional Model

Corneal Stromal Cell Plasticity: In Vitro Regulation of Cell Phenotype Through Cell-Cell Interactions in a Three-Dimensional Model

Decellularized Human Cornea for Reconstructing the Corneal Epithelium and Anterior Stroma

Isolation and Transplantation of Corneal Endothelial Cell–Like Cells Derived from In-Vitro-Differentiated Human Embryonic Stem Cells

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