ORCID Profile
0000-0002-5387-4209
Current Organisation
Singapore Eye Research Institute
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Publisher: Association for Research in Vision and Ophthalmology (ARVO)
Date: 08-07-2013
Abstract: There is a lack of definitive cell surface markers to differentiate cultured human corneal endothelial cells (HCECs) from stromal fibroblasts, which could contaminate HCEC cultures. The aim of our study is to discover cell surface antigens on HCECs that can be used to identify and purify HCECs from stromal fibroblasts. RNA sequencing (RNA-seq) was used to find differentially overexpressed genes in HCECs and commercial antibodies against these overexpressed antigens were screened by immunofluorescence assay. Similarly, 242 commercial antibodies against cell-surface antigens also were screened. Selected antibodies were used to sort HCECs from stromal fibroblasts by fluorescence-activated cell sorting (FACS). Two monoclonal antibodies, anti-GPC4 and anti-CD200, were identified to stain HCECs specifically. FACS was used successfully to sort HCECs away from stromal fibroblasts. Recovery efficiency of HCECs after sorting using anti-GPC4 antibody was higher compared to anti-CD200 antibody, but purity of HCECs culture using either antibody was comparable. Taken together, the anti-GPC4 and anti-CD200 antibodies can be useful for purification and identification of HCECs in cultures containing stromal fibroblasts.
Publisher: Springer Science and Business Media LLC
Date: 23-02-2016
DOI: 10.1038/SREP21661
Abstract: Cell surface antigens are important targets for monoclonal antibodies, but they are often difficult to work with due to their association with the cell membrane. Phage display is a versatile technique that can be applied to generate binders against difficult targets. Here we used antibody phage display to isolate a binder for a rare and specialized cell, the human corneal endothelial cell. The human corneal endothelium is a medically important cell layer defects in this layer account for about half of all corneal transplants. Despite its importance, no specific antigens have been found to mark this cell type. By panning a phage library directly on human corneal endothelial cells, we isolated an antibody that bound to these cells and not the other types of corneal cells. Subsequently, we identified the antibody’s putative target to be CD166 by immunoprecipitation and mass spectrometry. This approach can be used to isolate antibodies against other poorly-characterized cell types, such as stem cells or cancer cells, without any prior knowledge of their discriminating markers.
Publisher: Wiley
Date: 17-02-2015
DOI: 10.1002/WSBM.1292
Abstract: Tissue-engineered therapies are being developed across virtually all fields of medicine. Some of these therapies are already in clinical use, while others are still in clinical trials or the experimental phase. Most initial studies in the evaluation of new therapies focus on demonstration of clinical efficacy. However, cost considerations or economic viability are just as important. Many tissue-engineered therapies have failed to be impactful because of shortcomings in economic competitiveness, rather than clinical efficacy. Furthermore, such economic viability studies should be performed early in the process of development, before significant investment has been made. Cost-minimization analysis combined with sensitivity analysis is a useful model for the economic evaluation of new tissue-engineered therapies. The analysis can be performed early in the development process, and can provide valuable information to guide further investment and research. The utility of the model is illustrated with the practical real-world ex le of tissue-engineered constructs for corneal endothelial transplantation. The authors have declared no conflicts of interest for this article.
Publisher: Oxford University Press (OUP)
Date: 09-2007
DOI: 10.1095/BIOLREPROD.106.055244
Abstract: The amnion is the inner of two membranes surrounding the fetus. That it arises from embryonic epiblast cells prior to gastrulation suggests that it may retain a reservoir of stem cells throughout pregnancy. We found that human amniotic epithelial cells (hAECs) harvested from term-delivered fetal membranes express mRNA and proteins present in human embryonic stem cells (hESCs), including POU domain, class 5, transcription factor 1 Nanog homeobox SRY-box 2 and stage-specific embryonic antigen-4. In keeping with possible stem cell-like activity, hAECs were also clonogenic, and primary hAEC cultures could be induced to differentiate into cardiomyocytic, myocytic, osteocytic, adipocytic (mesodermal), pancreatic, hepatic (endodermal), neural, and astrocytic (neuroectodermal) cells in vitro, as defined by phenotypic, mRNA expression, immunocytochemical, and/or ultrastructural characteristics. However, unlike hESCs, hAECs did not form teratomas upon transplantation into severe combined immunodeficiency mice testes. Last, using flow cytometry we have shown that only a very small proportion of primary hAECs contain class IA and class II human leukocyte antigens (HLAs), consistent with a low risk of tissue rejection. However, following differentiation into hepatic and pancreatic lineages, significant proportions of cells contained class IA, but not class II, HLAs. These observations suggest that the term amnion, an abundant and easily accessible tissue, may be a useful source of multipotent stem cells that possess a degree of immune privilege.
Publisher: Association for Research in Vision and Ophthalmology (ARVO)
Date: 27-09-2013
Abstract: To establish an animal model of congenital hereditary endothelial dystrophy (CHED) using Slc4a11 knockout (KO) mice and evaluate the abnormalities in the cornea and kidney. The Slc4a11 KO mouse model was generated by gene deletion. Corneal abnormalities were evaluated using slit-l photography, anterior segment optical coherence tomography (AS-OCT), immunohistochemistry, RT-PCR, corneal endothelial cell staining, and electron microscopy. The temporal corneal changes were also monitored. Histological and functional changes of the kidney were also evaluated. Successful knockout of the Slc4a11 gene was confirmed by immunohistochemistry and RT-PCR. Slit-l photography and AS-OCT showed progressive corneal edema. Increased corneal endothelial cell size with decreased corneal endothelial cell density was observed with increased age. Scanning electron microscopy also revealed progressive cell swelling and distortion of the hexagonal cell morphology with time. Transmission electron microscopy showed characteristic ultrastructural findings of CHED, including endothelial vacuolization, thickening of the Descemet membrane, disorganization of collagen fibril, deposition of amorphous material, and progression of these changes with age. Decreased urine osmolarity and electrolyte concentrations suggesting abnormality in water resorption were also detected. Our Slc4a11 KO mouse model successfully represents clinical manifestations of human CHED. We were able to show chronological corneal progression for the first time in a knockout mouse model as well as renal abnormalities.
Publisher: Elsevier BV
Date: 05-2014
DOI: 10.1016/J.ACTBIO.2014.01.015
Abstract: The human corneal endothelium plays an important role in maintaining corneal transparency. Human corneal endothelial cells have limited regenerative capability in vivo. Consequently, endothelial dysfunction can occur following corneal endothelial trauma or inherited diseases. To restore endothelial function, corneal transplantation is needed. However, there is a worldwide shortage of donor corneas, motivating the development of a tissue-engineered graft alternative using cultivated endothelial cells. To induce in vitro cell proliferation, much effort has been made to improve culture conditions and to mimic the native extracellular microenvironment. We incorporated topographical and biochemical cues in our in vitro culture of human corneal endothelial cell line B4G12 (HCEC-B4G12) and hypothesized that manipulation of the extracellular environment can modulate cell proliferation, morphometry and phenotype. The topographies tested were nanopillars, microwells and micropillars on polydimethylsiloxane, while the biochemical factors were extracellular matrix protein coatings of fibronectin-collagen I (FC), FNC® coating mix (FNC) and laminin-chondroitin sulfate (LC). Cellular morphometry, Na(+)/K(+)-ATPase and zona occludens 1 (ZO-1) gene and protein expression were analyzed 3days after cells had formed a confluent monolayer. The cell circularity on all patterns and coatings was above 0.78. On all coatings, cell area was the lowest on micropillars. The coefficient of variation (CV) of the cell area was the lowest on nanopillars with an LC coating. With an FC coating, micropillars induced a better cellular outcome as the cells had the greatest circularity, smallest cell area and highest Na(+)/K(+)-ATPase and ZO-1 gene and protein expression. With the LC coating, HCECs grown on nanopillars resulted in the lowest CV of the cell area and the highest ZO-1 gene expression. Thus, HCEC-B4G12 morphometry and phenotype can be improved using different topographical and biochemical cues.
Publisher: Cambridge University Press (CUP)
Date: 2015
DOI: 10.1017/ERM.2015.16
Abstract: Regenerative medicine using patient's own stem cells (SCs) to repair dysfunctional tissues is an attractive approach to complement surgical and pharmacological treatments for aging and degenerative disorders. Recently, dental SCs have drawn much attention owing to their accessibility, plasticity and applicability for regenerative use not only for dental, but also other body tissues. In ophthalmology, there has been increasing interest to differentiate dental pulp SC and periodontal ligament SC (PDLSC) towards ocular lineage. Both can commit to retinal fate expressing eye field transcription factors and generate rhodopsin-positive photoreceptor-like cells. This proposes a novel therapeutic alternative for retinal degeneration diseases. Moreover, as PDLSC shares similar cranial neural crest origin and proteoglycan secretion with corneal stromal keratoctyes and corneal endothelial cells, this offers the possibility of differentiating PDLSC to these corneal cell types. The advance could lead to a shift in the medical management of corneal opacities and endothelial disorders from highly invasive corneal transplantation using limited donor tissue to cell therapy utilizing autologous cells. This article provides an overview of dental SC research and the perspective of utilizing dental SCs for ocular regenerative medicine.
Publisher: Hindawi Limited
Date: 25-04-2017
DOI: 10.1002/TERM.2131
Abstract: Corneal endothelial disorders collectively represent a significant healthcare burden in most developed nations, and corneal transplantation is currently the only treatment available for patients with poor visual acuity and corneal blindness secondary to endothelial failure. Although vision in these patients can be restored by transplantation, the global demand for donor human corneas is far in excess of what can be provided for by eye banks around the world, and this deficit is set to increase with an ageing global population. As such, there has been a pressing need to explore novel and more sustainable options for the treatment of corneal endothelial diseases. In recent years, significant progress has been made not only in the development of corneal endothelial cell culture techniques, but also in the exploration of various translational strategies. Considered together, we are now much closer to attaining success in the treatment of corneal endothelial diseases via a cell-based, tissue-engineering approach. The aim of this review article is to provide an update of the translational issues currently facing human corneal endothelial cell therapy. Copyright © 2016 John Wiley & Sons, Ltd.
Publisher: Elsevier BV
Date: 03-2017
DOI: 10.1016/J.BIOMATERIALS.2016.12.026
Abstract: Naturally-bioactive hydrogels like gelatin provide favorable properties for tissue-engineering but lack sufficient mechanical strength for use as implantable tissue engineering substrates. Complex fabrication or multi-component additives can improve material strength, but often compromises other properties. Studies have shown gelatin methacrylate (GelMA) as a bioactive hydrogel with erse tissue growth applications. We hypothesize that, with suitable material modifications, GelMA could be employed for growth and implantation of tissue-engineered human corneal endothelial cell (HCEC) monolayer. Tissue-engineered HCEC monolayer could potentially be used to treat corneal blindness due to corneal endothelium dysfunction. Here, we exploited a sequential hybrid (physical followed by UV) crosslinking to create an improved material, named as GelMA+, with over 8-fold increase in mechanical strength as compared to regular GelMA. The presence of physical associations increased the subsequent UV-crosslinking efficiency resulting in robust materials able to withstand standard endothelium insertion surgical device loading. Favorable biodegradation kinetics were also measured in vitro and in vivo. We achieved hydrogels patterning with nano-scale resolution by use of oxygen impermeable st s that overcome the limitations of PDMS based molding processes. Primary HCEC monolayers grown on GelMA+ carrier patterned with pillars of optimal dimension demonstrated improved zona-occludin-1 expression, higher cell density and cell size homogeneity, which are indications of functionally-superior transplantable monolayers. The hybrid crosslinking and fabrication approach offers potential utility for development of implantable tissue-engineered cell-carrier constructs with enhanced bio-functional properties.
Publisher: Springer Science and Business Media LLC
Date: 09-09-2009
DOI: 10.1007/S12015-009-9090-0
Abstract: Discontinuous density gradients have been shown to be very efficient in sperm enrichment in assisted reproductive technologies (ART). Percoll [polyvinylpyrrolidone (PVP)-coated silica] which is a colloidal agent commonly used in such gradients is now confined to only research purposes in ART because its PVP component was considered toxic for clinical applications. The PVP component was replaced with silane instead and silane-coated colloidal silica marketed as Puresperm is currently being used for clinical sperm enhancement procedures in ART. The efficiency of these two agents has not been tested for human embryonic stem cell (hESC) and cancer cell research and clinical application. Herein we describe discontinuous density gradient protocols using PureSperm and Percoll for the separation and enrichment of hESCs and hepatocarcinoma cells from a mixed cell population. Trypan blue cell viability and CellTracker green cell counts are used to accurately quantify the extent of cell separation. We show that Puresperm provides good cell separation and enrichment with higher viable cell counts and greater number of fractions compared to Percoll. PureSperm being superior and safer compared to Percoll would be a better alternative for cell separation and enrichment in stem and cancer cell research and clinical applications.
Publisher: Hindawi Limited
Date: 2017
DOI: 10.1155/2017/6906139
Abstract: Purpose . We evaluated the efficacy and safety of a mechanical device, the P-chute, in corneal endothelium preservation during phacoemulsification in a rabbit model. Methods . Twenty-four rabbits were randomly assigned into 2 groups. One eye of each rabbit underwent phacoemulsification that simulated the removal of a dense nucleus, with or without the P-chute. Serial slit-l examinations, anterior segment optical coherence tomography (ASOCT) scans, and specular microscopy were performed. Three rabbits from each group were sacrificed on postoperative days (PODs) 1, 5, 7, and 14. Histological analysis of the corneas was performed. Results . There was a trend towards lesser endothelial cell loss for the P-chute group at POD1 (4.9% versus 12.5%, p = 0.53 ), POD5 (10.4% versus 12.2%, p = 0.77 ), and POD7 (10.5% versus 17.2%, p = 0.52 ). There was no significant difference in the corneal thickness ( p = 0.05 ) between the 2 groups. The insertion of the device was challenging. The use of the P-chute only added an extra 15% to the surgical time. Conclusions . There was a trend towards better endothelium preservation with the P-chute even though the results were not statistically significant. We believe that the device could be useful in certain surgical situations. Further work is needed to improve the device insertion.
Publisher: Hindawi Limited
Date: 2012
DOI: 10.1155/2012/601302
Abstract: The culture of human corneal endothelial cells (CECs) is critical for the development of suitable graft alternative on biodegradable material, specifically for endothelial keratoplasty, which can potentially alleviate the global shortage of transplant-grade donor corneas available. However, the propagation of slow proliferative CECs in vitro can be hindered by rapid growing stromal corneal fibroblasts (CSFs) that may be coisolated in some cases. The purpose of this study was to evaluate a strategy using magnetic cell separation (MACS) technique to deplete the contaminating CSFs from CEC cultures using antifibroblast magnetic microbeads. Separated “labeled” and “flow-through” cell fractions were collected separately, cultured, and morphologically assessed. Cells from the “flow-through” fraction displayed compact polygonal morphology and expressed Na + /K + ATPase indicative of corneal endothelial cells, whilst cells from the “labeled” fraction were mostly elongated and fibroblastic. A separation efficacy of 96.88% was observed. Hence, MACS technique can be useful in the depletion of contaminating CSFs from within a culture of CECs.
Publisher: Public Library of Science (PLoS)
Date: 02-07-2013
Publisher: Wiley
Date: 09-2009
Publisher: Wiley
Date: 04-10-2016
Abstract: A common indication for corneal transplantation, which is the most transplanted tissue, is a dysfunctional corneal endothelium due to Fuchs' endothelial dystrophy (FED). FED is diagnosed by the presence of in vivo pathological microtopography on the Descemet membrane, which is called corneal guttata. Minimally invasive corneal endothelial cell regenerative procedures such as endothelial cell injection therapy and Rho kinase inhibitor pharmacotherapy have been proposed as alternatives to conventional corneal transplantation for FED patients. However, the effect of guttata on monolayer reformation following such therapies is unknown and there is no equivalent in vitro or animal model to study monolayer reformation. Using a synthetic guttata FED disease model, the formation of the monolayer is investigated to evaluate the efficacy of both therapies. Results obtained suggest that guttata dimensions, density, and spacing greatly affect the fate of corneal endothelial cells in terms of migratory behavior and monolayer reformation. Densely packed synthetic guttata mimicking late-stage FED hinders monolayer reformation, while synthetic guttata of lower height and density show improved monolayer formation. These results suggest that severity of the FED, as determined by height and density of existing guttata, can potentially attenuate corneal endothelial monolayer formation of corneal cell injection therapy and pharmacotherapy.
Publisher: Association for Research in Vision and Ophthalmology (ARVO)
Date: 04-12-2014
Abstract: To investigate the quality of the ultrathin corneal grafts prepared by femtosecond laser from the endothelial side for Descemet stripping endothelial keratoplasty. Thirty human corneoscleral buttons were cut from the endothelial side by laser Doppler velocimetry (LDV) with or without viscoelastic materials coating. Two cutting depths were selected: 70 and 90 μm. The postcut endothelial count was determined by specular microscopy, and the graft thickness was evaluated by anterior segment optical coherence tomography. The endothelial viability was determined using Trypan blue/Alizarin red staining, calcein-AM/EthD-1 live/dead cell assay, and scanning electron microscope (SEM). The graft interface smoothness was evaluated by SEM. Another 18 corneoscleral buttons were used as controls for the comparisons. The overall targeted cutting depth and achieved cutting depth were significantly highly correlated (r = 0.84). The central to peripheral corneal thickness ratio was 0.976 and 0.998 for the 70- and 90-μm grafts. The percentage of the damaged endothelial cells assessed by vital staining and SEM showed the 70-μm grafts had noticeably more endothelial damage compared with the 90-μm grafts. But the damage was significantly reduced, to the control corneas level, after coating the endothelium with Viscoat. The 90-μm grafts had a slightly rougher graft interface than the 70-μm grafts, but all the grafts dissected by a Chansue dissector exhibited a generally smooth interface. The corneal endothelial grafts prepared by LDV femtosecond laser with endothelial approach produced consistently ultrathin grafts in uniform shape with high accuracy and good endothelial and stromal interface quality.
Publisher: Public Library of Science (PLoS)
Date: 16-12-2011
Publisher: Public Library of Science (PLoS)
Date: 30-11-2012
Publisher: Association for Research in Vision and Ophthalmology (ARVO)
Date: 08-09-2017
Abstract: To characterize the differences in corneal endothelial wound healing in the presence or absence of Descemet's membrane (DM), in vivo. New-Zealand white rabbits were subjected to 7-mm endothelial wound either by scraping (n = 8 DM intact), peeling (n = 6 DM removed), or a combinatory scrape eel wound (n = 6). In a second experiment, rabbits underwent peel wound with immediate transplantation of pure decellularized human DM (n = 4). In vivo endothelial migration was assessed via trypan blue staining. Recovery of corneal clarity and thickness was performed by using slit-l biomicroscopy and optical coherence tomography. Cell proliferation, phenotype, and morphology were assessed by using immunofluorescence and scanning electron microscopy. In vivo wound closure was faster in the presence of DM 25.4% ± 1.4%/d versus 5.5% ± 0.6%/d (P < 0.0001). At day 8, complete wound closure was seen in all of the scrape s les but none of the peel group, with wound closure preceding clinical recovery by approximately 6 days in the scrape group. Endothelial cells in the scraped areas reformed functional monolayers capable of restoring corneal thickness and transparency whilst those in the peeled area underwent mesenchymal-like transformation resulting in scar formation. Transplanting decellularized DM in animals receiving a peel wound resulted in clarity and thickness comparable to the scrape group. Endothelial proliferation (Ki67 +ve cells) was higher in scraped versus peeled areas: 54.7% ± 3.5% vs. 8.8% ± 0.7%, (P < 0.01). The presence of DM promoted endothelial wound healing, proliferation, and maintenance of a normal phenotype. DM transplantation recovered the abnormal peel phenotype back to that observed after endothelial scraping.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 27-04-2011
Publisher: Springer Science and Business Media LLC
Date: 31-03-2016
DOI: 10.1038/SREP23836
Abstract: Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4 th _FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4 th _FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities.
Publisher: MDPI AG
Date: 03-05-2023
Abstract: (1) Rho-associated coiled-coil protein kinase (ROCK) signaling cascade impacts a wide array of cellular events. For cellular therapeutics, scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and the inhibition of ROCK signaling using a well characterized ROCK inhibitor (ROCKi) Y-27632 had been shown to enhance overall endothelial cell yield. (2) In this study, we compared several classes of ROCK inhibitors to both ROCK-I and ROCK-II, using in silico binding simulation. We then evaluated nine ROCK inhibitors for their effects on primary CECs, before narrowing it down to the two most efficacious compounds—AR-13324 (Netarsudil) and its active metabolite, AR-13503—and assessed their impact on cellular proliferation in vitro. Finally, we evaluated the use of AR-13324 on the regenerative capacity of donor cornea with an ex vivo corneal wound closure model. Donor-matched control groups supplemented with Y-27632 were used for comparative analyses. (3) Our in silico simulation revealed that most of the compounds had stronger binding strength than Y-27632. Most of the nine ROCK inhibitors assessed worked within the concentrations of between 100 nM to 30 µM, with comparable adherence to that of Y-27632. Of note, both AR-13324 and AR-13503 showed better cellular adherence when compared to Y-27632. Similarly, the proliferation rates of CECs exposed to AR-13324 were comparable to those of Y-27632. Interestingly, CECs expanded in a medium supplemented with AR-13503 were significantly more proliferative in (i) untreated vs. AR-13503 (1 μM * p 0.05) (ii) untreated vs. AR-13503 (10 μM *** p 0.001) (iii) Y-27632 vs. AR-13503 (10 μM ** p 0.005) (iv) AR-13324 (1 μM) vs. AR-13503 (10 μM ** p 0.005) and (v) AR-13324 (0.1 μM) vs. AR-13503 (10 μM * p 0.05). Lastly, an ex vivo corneal wound healing study showed a comparable wound healing rate for the final healed area in corneas exposed to Y-27632 or AR-13324. (4) In conclusion, we were able to demonstrate that various classes of ROCKi compounds other than Y-27632 were able to exert positive effects on primary CECs, and systematic donor-match controlled comparisons revealed that the FDA-approved ROCK inhibitor, AR-13324, is a potential candidate for cellular therapeutics or as an adjunct drug in regenerative treatment for corneal endothelial diseases in humans.
Publisher: Springer Science and Business Media LLC
Date: 26-10-2017
DOI: 10.1038/S41598-017-14723-Z
Abstract: Corneal transplantation is the only treatment available to restore vision for in iduals with blindness due to corneal endothelial dysfunction. However, severe shortage of available donor corneas remains a global challenge. Functional regulatory compliant tissue-engineered corneal endothelial graft substitute can alleviate this reliance on cadaveric corneal graft material. Here, isolated primary human corneal endothelial cells (CEnCs) propagated using a dual media approach refined towards regulatory compliance showed expression of markers indicative of the human corneal endothelium, and can be tissue-engineered onto thin corneal stromal carriers. Both cellular function and clinical adaptability was demonstrated in a pre-clinical rabbit model of bullous keratopathy using a tissue-engineered endothelial keratoplasty (TE-EK) approach, adapted from routine endothelial keratoplasty procedure for corneal transplantation in human patients. Cornea thickness of rabbits receiving TE-EK graft gradually reduced over the first two weeks, and completely recovered to a thickness of approximately 400 µm by the third week of transplantation, whereas corneas of control rabbits remained significantly thicker over 1,000 µm ( p 0.05) throughout the course of the study. This study showed convincing evidence of the adaptability of the propagated CEnCs and their functionality via a TE-EK approach, which holds great promises in translating the use of cultured CEnCs into the clinic.
Publisher: Mary Ann Liebert Inc
Date: 03-2009
Abstract: The ability to generate purified neural progenitors is critical to the development of embryonic stem cell-based therapies to alleviate human neurological disorders. While many cell culture protocols for directed differentiation of human embryonic stem (hES) cells into neural cells have been described, most yield mixed populations, some containing cells of different embryonic germ layer lineages, or even undifferentiated embryonic stem cells. In this study, we describe a method for single-cell dissociation, isolation by flow cytometry, and subsequent culture of neural progenitors from hES cells. As reported earlier, hES cells treated with the bone morphogenetic protein (BMP) antagonist noggin gave rise to neurospheres at a relatively high frequency. However, these noggin-treated embryonic stem cell cultures were heterogeneous, with cells expressing embryonic stem markers still detectable even following 14 days of differentiation. In order to isolate pure human neural progenitors, we fractionated noggin-treated ES cells on the basis of their expression of the putative neural stem cell marker, CD133, and the GCTM-2, and SSEA-1 antigens, which mark pluripotent stem cells and differentiated cells respectively from hES cell culture. CD133(+) cells formed larger spheres compared to CD133(-) cells. CD133(+)SSEA1(+) cells and CD133(+)SSEA-1(-) cells expressed similar levels of neural genes and formed neurospheres at similar frequencies. By contrast, CD133(+)GCTM-2(+) cells expressed high levels of OCT4 but not neural lineage genes and failed to form neurospheres. CD133(+)GCTM-2(-) cells formed neurospheres at the relative highest frequency. Thus, negative selection with GCTM-2 may be useful for the purification of specific cell types differentiated from hES cells.
Publisher: Association for Research in Vision and Ophthalmology (ARVO)
Date: 03-02-2016
Abstract: To characterize the effects of Descemet's stripping, Rho-associated protein kinase inhibitor Y-27632, and donor age on endothelial migration in human corneas maintained in ex vivo culture. Twenty-eight cadaveric human corneas underwent ex vivo culture in either standard or Y-27632-supplemented culture medium for 14 days. The posterior surface of each cornea was manipulated to create two types of wounds: scratched wound--corneal endothelial cells (CECs) were denuded from the Descemet's membrane (DM) to leave behind a bare but intact DM and peeled wound--both the DM and overlying CECs were stripped to leave behind bare corneal stroma. Endothelial migration was assessed via Trypan blue staining. Morphologic traits of CECs were assessed via Alizarin red microscopy and scanning electron microscopy. The CECs migrated preferentially over scratched wounds compared with peeled wounds. Y-27632 supplementation accelerated endothelial migration over scratched wounds. Endothelial migration decreased with advanced donor age for both wound types, regardless of exposure to Y-27632. Y-27632 supplementation resulted in a less rapid decline in endothelial migration for donors older than 50 years of age for scratched surfaces. Greater cell density and hexagonality was observed over scratched wounds compared with peeled wounds, regardless of Y-27632 supplementation. The presence of an intact DM, Y-27632 supplementation, and young donor age are factors that promote endothelial migration in an ex vivo human cornea culture model. The negative effect of age on endothelial migration can be mitigated by the presence of an intact DM and Y-27632 supplementation.
Publisher: Elsevier BV
Date: 06-2015
DOI: 10.1016/J.ACTBIO.2015.03.016
Abstract: One of the most common indications for corneal transplantation is corneal endothelium dysfunction, which can lead to corneal blindness. Due to a worldwide donor cornea shortage, alternative treatments are needed, but the development of new treatment strategies relies on the successful in vitro culture of primary human corneal endothelial cells (HCECs) because transformed cell lines and animal-derived corneal endothelial cells are not desirable for therapeutic applications. Primary HCECs are non-proliferative in vivo and challenging to expand in vitro while maintaining their characteristic cell morphology and critical markers. Biochemical cues such as growth factors and small molecules have been investigated to enhance the expansion of HCECs with a limited increase in proliferation. In this study, patterned tissue culture polystyrene (TCPS) was shown to significantly enhance the expansion of HCECs. The proliferation of HCECs increased up to 2.9-fold, and the expression amount and localization of cell-cell tight junction protein Zona Occludens-1 (ZO-1) was significantly enhanced when grown on 1 μm TCPS pillars. 250 nm pillars induced an optimal hexagonal morphology of HCEC cells. Furthermore, we demonstrated that the topographical effect on tight-junction expression and cell morphology could be maintained throughout each passage, and was effectively 'remembered' by the cells. Higher amount of tight-junction protein expression was maintained at cell junctions when topographic cues were removed in the successive seeding. This topographic memory suggested topography-exposed/induced cells would maintain the enhanced functional markers, which would be useful in cell-therapy based approaches to enable the in situ endothelial cell monolayer formation upon delivery. The development of patterned TCPS culture platforms could significantly benefit those researching human corneal endothelial cell cultivation for cell therapy, and tissue engineering applications.
Publisher: Portland Press Ltd.
Date: 09-05-2017
DOI: 10.1042/BCJ20170125
Abstract: Corneal stromal dystrophies are a group of genetic disorders that may be caused by mutations in the transforming growth factor β-induced (TGFBI) gene which results in the aggregation and deposition of mutant proteins in various layers of the cornea. The type of amino acid substitution dictates the age of onset, anatomical location of the deposits, morphological features of deposits (amyloid, amorphous powder or a mixture of both forms) and the severity of disease presentation. It has been suggested that abnormal turnover and aberrant proteolytic processing of the mutant proteins result in the accumulation of insoluble protein deposits. Using mass spectrometry, we identified increased abundance of a 32 amino acid-long peptide in the 4th fasciclin-like domain-1 (FAS-1) domain of transforming growth factor β-induced protein (amino acid 611–642) in the amyloid deposits of the patients with lattice corneal dystrophies (LCD). In vitro studies demonstrated that the peptide readily formed amyloid fibrils under physiological conditions. Clinically relevant substitution (M619K, N622K, N622H, G623R and H626R) of the truncated peptide resulted in profound changes in the kinetics of amyloid formation, thermal stability of the amyloid fibrils and cytotoxicity of fibrillar aggregates, depending on the position and the type of the amino acid substitution. The results suggest that reduction in the overall net charge, nature and position of cationic residue substitution determines the amyloid aggregation propensity and thermal stability of amyloid fibrils.
Publisher: Future Medicine Ltd
Date: 09-2016
Abstract: Corneal integrity is essential for visual function. Transplantation remains the most common treatment option for advanced corneal diseases. A global donor material shortage requires a search for alternative treatments. Different stem cell populations have been induced to express corneal cell characteristics in vitro and in animal models. Yet before their application to humans, scientific and ethical issues need to be solved. The in vitro propagation and implantation of primary corneal cells has been rapidly evolving with clinical practices of limbal epithelium transplantation and a clinical trial for endothelial cells in progress, implying cultivated ocular cells as a promising option for the future. This review reports on the latest developments in primary ocular cell and stem cell research for corneal therapy.
Publisher: Springer Science and Business Media LLC
Date: 31-01-2009
DOI: 10.1007/S12015-009-9054-4
Abstract: A major concern in human embryonic stem cell (hESC)-derived cell replacement therapy is the risk of tumorigenesis from undifferentiated hESCs residing in the population of hESC-derived cells. Separation of these undifferentiated hESCs from the differentiated derivatives using cell sorting methods may be a plausible approach in overcoming this problem. We therefore explored magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) to separate labelled undifferentiated hESCs from a heterogeneous population of hESCs and hepatocellular carcinoma cells (HepG2) deliberately mixed respectively at different ratios (10:90, 20:80, 30:70, 40:60 and 50:50) to mimic a standard in vitro differentiation protocol, instead of using a hESC-differentiated cell population, so that we could be sure of the actual number of cells separated. HES-3 and HES-4 cells were labelled in separate experiments for the stem cell markers SSEA-4 and TRA-1-60 using primary antibodies. Anti-PE magnetic microbeads that recognize the PE-conjugated SSEA-4 labelled hESCs was added to the heterogeneous cell mixture and passed through the MACS column. The cells that passed through the column ('flow-through' fraction) and those retained ('labelled' fraction') were subsequently analysed using FACS. The maximum efficacy of hESCs retention using MACS was 81.0 +/- 2.9% (HES-3) and 83.6 +/- 4.2% (HES-4). Using FACS, all the undifferentiated hESCs labelled with the two cell-surface markers could be removed by selective gating. Both hESCs and HepG2 cells in the 'flow-through' fraction following MACS separation were viable in culture whereas by FACS separation only the HepG2 cells were viable. FACS efficiently helps to eliminate the undifferentiated hESCs based on their cell-surface antigens expressed.
Publisher: Association for Research in Vision and Ophthalmology (ARVO)
Date: 30-04-2013
Abstract: We characterized the de-epithelialized human amniotic membrane (HAM), and compared cell attachment and proliferation efficiencies. HAM was de-epithelialized by 20% ethanol (AHAM), 1.2 U/mL Dispase (DHAM), 0.02% EDTA (EHAM), 0.25% trypsin-EDTA (THAM), and 5 M urea (UHAM), respectively, followed by gentle scraping with a #15 blade. Surface topology, extracellular matrix (ECM), and growth factor content were characterized and compared to intact HAM by electron microscopies (EM), atomic force microscopy (AFM), immunohistochemistry, and Western blotting. Primary human limbal epithelial cells (LEC) attachment and proliferation efficiencies were assayed. Statistical significance was calculated by SPSS and Fisher's least significant difference test. EHAM, THAM, and UHAM had intact basal lamina and smooth basement membrane surface shown under transmission and scanning EM, and AFM. Cell remnants stayed on AHAM. Disrupted basement membrane and stroma was found in DHAM. Immunostaining intensity quantification and hierarchical clustering revealed that ECM composition of EHAM and UHAM resembled intact HAM. In contrast, DHAM and THAM had drastic loss of ECM and growth factor content. LEC attachment efficiency at 24 hours after seeding was the highest in EHAM (51% as on conventional culture surface), followed by UHAM and AHAM. However, cell proliferation indices at day 10 of culture were similar among different HAM substrates, suggesting repair of ECM and basement membrane by growing epithelial cells. Urea denudation preserved the basement membrane integrity, ECM, and growth factor composition, and had higher cell attachment and proliferation efficiencies. With its short processing time, urea treatment offers a novel alternative for HAM de-epithelialization.
Publisher: Springer Science and Business Media LLC
Date: 06-10-2015
DOI: 10.1038/SREP14742
Abstract: Cataract surgery is the most common surgical procedure and femtosecond laser assisted cataract surgery (FLACS) has gained increased popularity. FLACS requires the application of a suction device to stabilize the laser head and focus the laser beam accurately. This may cause a significant escalation in intra-ocular pressure (IOP), which poses potential risks for patients undergoing cataract surgery. In this study we aimed to assess the effect of the Ziemer LDV Z8 femtosecond cataract machine on IOP. We demonstrated through a porcine model that IOP was significantly higher with a flat interface but could be abrogated by reducing surgical compression and vacuum. Pressure was lower with a liquid interface and further altering angulation of the laser arm could reduce the IOP to 36 mmHg. A pilot series in patients showed comparable pressure rises with the porcine model (30 mmHg). These strategies may improve the safety profile in patients vulnerable to high pressure when employing FLACS with the Ziemer LDV Z8.
Publisher: Oxford University Press (OUP)
Date: 11-2005
DOI: 10.1634/STEMCELLS.2004-0338
Abstract: Human embryonic stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the factors that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum--sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF)--in maintaining hESCs. We show here that although LPA does not affect hESC growth or differentiation, coincubation of S1P and PDGF in a serum-free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri-potentiality.
Publisher: Springer Science and Business Media LLC
Date: 15-02-2017
DOI: 10.1007/S00347-017-0454-6
Abstract: In recent years, the cultivation and expansion of primary corneal cells has made significant progress. The transplantation of cultured limbal epithelial cells represents a successful and established treatment of the ocular surface. Cultivated corneal endothelial cells are undergoing a clinical trial in Japan. Stromal keratocytes can now be expanded in vitro. A wide range of stem cell sources is being tested in vitro and animal models for their possible application in corneal cell therapy. This article gives an overview of recent advancements and prevailing limitations for the use of different cell sources in the therapy of corneal disease.
Publisher: Springer Science and Business Media LLC
Date: 16-03-2015
DOI: 10.1038/SREP09167
Abstract: The global shortage of donor corneas has garnered extensive interest in the development of graft alternatives suitable for endothelial keratoplasty using cultivated primary human corneal endothelial cells (CECs). We have recently described a dual media approach for the propagation of human CECs. In this work, we characterize the effects of a Rho-kinase inhibitor Y-27632 on the cultivation of CECs propagated using the dual media culture system. Seventy donor corneas deemed unsuitable for transplantation were procured for this study. We assessed the use of Y-27632 for its effect at each stage of the cell culture process, specifically for cell attachment, cell proliferation and during both regular passaging and cryopreservation. Lastly, comparison of donor-matched CEC-cultures expanded with or without Y-27632 was also performed. Our results showed that Y-27632 significantly improved the attachment and proliferation of primary CECs. A non-significant pro-survival effect was detected during regular cellular passage when CECs were pre-treated with Y-27632, an effect that became more evident during cryopreservation. Our study showed that the inclusion of Y-27632 was beneficial for the propagation of primary CECs expanded via the dual media approach and was able to increase overall cell yield by between 1.96 to 3.36 fold.
Publisher: Springer Science and Business Media LLC
Date: 15-04-2019
DOI: 10.1038/S41598-019-42493-3
Abstract: Restoration of vision due to corneal blindness from corneal endothelial dysfunction can be achieved via a corneal transplantation. However, global shortage of donor tissues has driven the development cell-based therapeutics. With the capacity to propagate regulatory compliant human corneal endothelial cells (CEnCs), this study evaluated the functionality of propagated CEnCs delivered via tissue-engineered endothelial keratoplasty (TE-EK) or corneal endothelial cell injection (CE-CI) within a rabbit model of bullous keratopathy. For animals with TE-EK grafts, central corneal thickness (CCT) increased to μm post-operatively. Gradual thinning with improvements in corneal clarity was observed from week 1. CCT at week 3 was 484.3 ± 73.7 μm. In rabbits with CE-CI, corneal clarity was maintained throughout, and CCT at week 3 was 582.5 ± 171.5 μm. Control corneas remained significantly edematous throughout the study period compared to their respective experimental groups ( p 0.05). Characterization of excised corneas showed a monolayer with heterogeneously shaped CEnCs in both TE-EK and CE-CI groups. Immunohistochemistry demonstrated reactivity to anti-human specific nuclei antibody attributing corneal recovery to the functional human CEnCs. This study showed that regulatory compliant cell-based therapy for corneal endothelial dysfunction can be delivered by both TE-EK and CE-CI, and holds great promise as an alternative to traditional corneal transplantation.
Publisher: Springer Science and Business Media LLC
Date: 30-03-2023
DOI: 10.1038/S42003-023-04725-1
Abstract: TGFBI-related corneal dystrophy (CD) is characterized by the accumulation of insoluble protein deposits in the corneal tissues, eventually leading to progressive corneal opacity. Here we show that ATP-independent amyloid-β chaperone L-PGDS can effectively disaggregate corneal amyloids in surgically excised human cornea of TGFBI-CD patients and release trapped amyloid hallmark proteins. Since the mechanism of amyloid disassembly by ATP-independent chaperones is unknown, we reconstructed atomic models of the amyloids self-assembled from TGFBIp-derived peptides and their complex with L-PGDS using cryo-EM and NMR. We show that L-PGDS specifically recognizes structurally frustrated regions in the amyloids and releases those frustrations. The released free energy increases the chaperone’s binding affinity to amyloids, resulting in local restructuring and breakage of amyloids to protofibrils. Our mechanistic model provides insights into the alternative source of energy utilized by ATP-independent disaggregases and highlights the possibility of using these chaperones as treatment strategies for different types of amyloid-related diseases.
Publisher: SAGE Publications
Date: 02-2015
Abstract: Corneal endothelium-associated corneal blindness is the most common indication for corneal transplantation. Restorative corneal transplant surgery is the only option to reverse the blindness, but a global shortage of donor material remains an issue. There are immense clinical interests in the development of alternative treatment strategies to alleviate current reliance on donor materials. For such endeavors, ex vivo propagation of human corneal endothelial cells (hCECs) is required, but current methodology lacks consistency, with expanded hCECs losing cellular morphology to a mesenchymal-like transformation. In this study, we describe a novel dual media culture approach for the in vitro expansion of primary hCECs. Initial characterization included analysis of growth dynamics of hCECs grown in either proliferative (M4) or maintenance (M5) medium. Subsequent comparisons were performed on isolated hCECs cultured in M4 alone against cells expanded using the dual media approach. Further characterizations were performed using immunocytochemistry, quantitative real-time PCR, and gene expression microarray. At the third passage, results showed that hCECs propagated using the dual media approach were homogeneous in appearance, retained their unique polygonal cellular morphology, and expressed higher levels of corneal endothelium-associated markers in comparison to hCECs cultured in M4 alone, which were heterogeneous and fibroblastic in appearance. Finally, for hCECs cultured using the dual media approach, global gene expression and pathway analysis between confluent hCECs before and after 7-day exposure to M5 exhibited differential gene expression associated predominately with cell proliferation and wound healing. These findings showed that the propagation of primary hCECs using the novel dual media approach presented in this study is a consistent method to obtain bona fide hCECs. This, in turn, will elicit greater confidence in facilitating downstream development of alternative corneal endothelium replacement using tissue-engineered graft materials or cell injection therapy.
Publisher: Public Library of Science (PLoS)
Date: 04-10-2017
Publisher: Springer Science and Business Media LLC
Date: 03-05-2013
Abstract: Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology. Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm 2 (‘LOW’) 5,000 cells per cm 2 (‘MID’) 10,000 cells per cm 2 (‘HIGH’) and 20,000 cells per cm 2 (‘HIGH ×2 ’), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm 2 , it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology. Our results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm 2 for regular passage of primary human corneal endothelial cells.
Publisher: Public Library of Science (PLoS)
Date: 20-06-2014
No related grants have been discovered for Gary Peh.