Substrates for Expansion of Corneal Endothelial Cells towards Bioengineering of Human Corneal Endothelium

Navaratnam, Jesintha; Utheim, Tor Paaske; Rajasekhar, Vinagolu K.; Shahdadfar, Aboulghassem

doi:10.3390/jfb6030917

Cited by 51 publications

(57 citation statements)

References 137 publications

(233 reference statements)

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“…Transplanting the LC with the epithelial side down resulted in a more rapid recovery and a transparent cornea at 6 months when compared with control eyes. LC has also been used successfully as a scaffold for the expansion of both limbal epithelial stem cells and HCEnC in vitro (Kopsachilis et al, ; Lachaud et al, ; Navaratnam et al, ; Yoeruek et al, ). These data suggested that LC could eventually be used for human transplantation, but prior to in vivo use, further characterization is required.…”

Section: Discussionmentioning

confidence: 99%

“…The two delivery systems have been described, namely, (a) an intracameral injectable endothelial cell suspension, or (b) tissue‐engineered endothelial grafts utilizing a carrier membrane or scaffold. Culturing the cells on a scaffold resembles the current endothelial surgical techniques, and as such, the majority of research groups involved in this domain are investigating the scaffold‐based approach (Navaratnam, Utheim, Rajasekhar, & Shahdadfar, ).…”

Section: Introductionmentioning

confidence: 99%

“…Corneal scaffolds must also be avascular and permeable to the aqueous, which supplies nutrients to the posterior corneal stroma. Lastly, the scaffold should also be cytocompatible and favour cellular adhesion and proliferation (Navaratnam et al, ).…”

Section: Introductionmentioning

confidence: 99%

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Characterizing human decellularized crystalline lens capsules as a scaffold for corneal endothelial tissue engineering

Bogerd

Dhubhghaill

Zakaria

2018

J Tissue Eng Regen Med

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The idea of transplanting a sheet of laboratory‐grown corneal endothelium dates back to 1978; however, the ideal scaffold is still lacking. We hypothesized that human crystalline lens capsules (LCs) could qualify as a scaffold and aimed to characterize the properties of this material for endothelial tissue engineering. LCs were isolated from donor eyes, stored at −80 °C, and decellularized with water and trypsin‐EDTA. The decellularization was investigated by nuclear staining and counting and the capsule thickness was determined by optical coherence tomography and compared with Descemet's membrane (DM). Transparency was examined by spectrometry, and collagenase degradation was performed to evaluate its resistance to degradation. Cell‐scaffold interaction was assessed by measuring focal adhesions surface area on LC and plastic. Finally, primary corneal endothelial cells were grown on LCs to validate the phenotype. Trypsin‐EDTA decellularized most effectively, removing 99% of cells. The mean LC thickness was 35.76 ± 0.43 μm, whereas DM measured 25.93 ± 0.26 μm (p < .0001). Light transmission was 90% for both LC and DM. On a collagenase challenge, LC and amniotic membrane were digested after 13 hr, whereas DM was digested after 17 hr. The surface area of focal adhesions for cells grown on coated LCs was at least double that compared with other conditions, whereas tight junctions, ion pumps, and hexagonal morphology were well maintained when endothelial cells were cultured on LCs. In conclusion, LCs demonstrate excellent scaffolding properties for tissue engineering and sustain the cell phenotype and can be considered a suitable substrate for ocular tissue engineering or as a template for future scaffolds.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterizing human decellularized crystalline lens capsules as a scaffold for corneal endothelial tissue engineering

Bogerd

Dhubhghaill

Zakaria

2018

J Tissue Eng Regen Med

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“…The corneal endothelium layer maintains corneal transparency by balancing its hydration through barrier and pump functions [1,2]. Damage of the hexagonal-shaped corneal endothelial cells (CECs) can cause severe visual impairment and can even lead to blindness [3].…”

Section: Introductionmentioning

confidence: 99%

LY2109761, Transforming Growth Factor β Receptor Type I and Type II Dual Inhibitor, is a Novel Approach to Suppress Endothelial Mesenchymal Transformation in Human Corneal Endothelial Cells

Zhang¹,

Miao²,

Ke³

et al. 2018

Cell Physiol Biochem

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Background/Aims: Preventing undesirable endothelial-mesenchymal transformation (EnMT) with repetitious in vitro expansion of human corneal endothelial cells (CECs) is a pivotal issue in cornea regeneration. Previous studies have shown that inhibition of the TGF-β pathway reduces epithelial-mesenchymal transformation. However, its potential role in EnMT remains poorly understood. As such, the effect of LY2109761, a novel TGF-β receptor type I and type II dual inhibitor, was investigated on EnMT. Methods: CECs cultured with various concentrations of LY2109761 were evaluated for their growth rate and phenotype. Additionally, the expression of functional markers (sodium-potassium pump Na⁺/K⁺-ATPase and the tight junction protein ZO-1) and mesenchymal markers (CD73, fibronectin, and vimentin) was detected using immunostaining and western blot. The mRNA expressions were also assayed by real-time polymerase chain reaction analysis. Results: At a 1 μM concentration, LY2109761 did not influence the proliferation of CECs and subsequent experiments were therefore performed using this concentration. Furthermore, CECs cultured in the presence of 1 μM LY2109761 maintained their ability to grow as a monolayer of hexagonal-shaped cells. The expression of functional markers increased in LY2109761-treated CECs, while the expression of mesenchymal markers decreased (both in protein and mRNA levels). Conclusion: Inhibition of TGF-β receptor type I and type II by LY2109761 maintained the phenotype of CECs and inhibited the EnMT process. These results indicate the possible continuous in vitro expansion of CECs with normal function.

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“…32 Meanwhile, mature CECs can also provide some inducing factors for undifferentiated CECs by autocrining some regulatory factors. 33,34 At this stage, we induced the differentiation of POMPs using a mixed-conditioned medium of both LEC-CM and HCEC-CM. The morphologic changes of cells were documented by light microscopy (Fig.…”

Section: The Isolation and Characterization Of Cec-like Cellsmentioning

confidence: 99%

Directed Differentiation of Human Corneal Endothelial Cells From Human Embryonic Stem Cells by Using Cell-Conditioned Culture Media

Chen

et al. 2018

Invest. Ophthalmol. Vis. Sci.

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Citation: Chen X, Wu L, Li Z, et al. Directed differentiation of human corneal endothelial cells from human embryonic stem cells by using cellconditioned culture media. Invest Ophthalmol Vis Sci. 2018;59:3028-3036. https://doi.org/10.1167/ iovs.17-23627 PURPOSE. A shortage of human corneal endothelial cells (HCEC) for transplant and current methods of differentiation induction require chemical compounds, which might cast further influences after differentiation induction. Therefore, we developed a simple and straightforward approach to endothelial cell differentiation from human embryonic stem cells (hESC).METHODS. HESC are used to differentiate into HCEC by employing a two-stage method, which involves the application of two different types of conditioned culture medium, human corneal stromal cell-conditioned medium (HCSC-CM) and lens epithelial cell (LEC) plus HCSC-CM (LEC-CMþHCEC-CM). In brief, hESCs were treated with different conditioned media to induce directed endothelial differentiation. RESULTS.In the presence of conditioned culture medium, embryonic stem cells differentiate first under the control of periocular mesenchymal precursors (POMPs). Consequently, the expression of several POMP markers was observed. Following this first stage differentiation, POMPs were further directed to differentiate into corneal endothelial cell (CEC)-like cells in the presence of the second-conditioned culture medium. The differentiation of POMPs into CEC-like cells is regulated by a TGFb-2/FOXC1 signaling pathway that is activated by the factors present in the conditioned culture medium.CONCLUSIONS. HCEC-like cells could be differentiated from hESC by simply using a two-step, preconditioned, medium-mediated approach, which could significantly minimize the workload to generate HCEC for potential clinical use. This research may provide an ideal cell source for corneal regenerative medicine and clinical treatment for corneal diseases in the future.

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Substrates for Expansion of Corneal Endothelial Cells towards Bioengineering of Human Corneal Endothelium

Cited by 51 publications

References 137 publications

Characterizing human decellularized crystalline lens capsules as a scaffold for corneal endothelial tissue engineering

Characterizing human decellularized crystalline lens capsules as a scaffold for corneal endothelial tissue engineering

LY2109761, Transforming Growth Factor β Receptor Type I and Type II Dual Inhibitor, is a Novel Approach to Suppress Endothelial Mesenchymal Transformation in Human Corneal Endothelial Cells

Directed Differentiation of Human Corneal Endothelial Cells From Human Embryonic Stem Cells by Using Cell-Conditioned Culture Media

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