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1.  Wnt Signaling Induces Epithelial-Mesenchymal Transition with Proliferation in ARPE-19 Cells upon Loss of Contact Inhibition 
Proliferation and epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) are hallmarks of proliferative vitreoretinopathy. This study aims at clarifying the role of growth factors, i.e. epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), and transforming growth factor-β1 (TGF-β1) in controlling how RPE proliferates while undergoing EMT. When contact inhibition of post-confluent ARPE-19 cells was disrupted by EGTA, an increase of BrdU labeling was noted only in the presence of EGF and/or FGF-2, and was accompanied by EMT as evidenced by the loss of a normal RPE phenotype (altered cytolocalization of RPE65, N-cadherin, ZO-1, and Na,K-ATPase) and the gain of a mesenchymal phenotype (increased expression of vimentin, S100A4, and α-SMA). EMT with proliferation by EGTA plus EGF+FGF-2 was accompanied by activation of canonical Wnt signaling (judged by the TCF/LEF promoter activity, increased nuclear levels of and interaction between β-catenin and LEF1 proteins, and the replication by overexpression of β-catenin), abolished by concomitant addition of XAV939, a Wnt inhibitor, but not associated with suppression of Hippo signaling (negative expression of nuclear TAZ or YAP and cytoplasmic p-TAZ or p-YAP). The causative role of Wnt signaling on EMT with proliferation was confirmed by overexpression of stable S33Y β-catenin with EGTA treatment. In addition, contact inhibition disrupted by EGTA in the presence of TGF-β1 also led to EMT but suppressed proliferation and Wnt signaling. The Wnt signaling triggered by EGF+FGF-2 was sufficient and synergized with TGF-β1 in activating the Smad/ZEB1/2 signaling responsible for EMT. These findings establish a framework for further dissecting how RPE might partake in a number of proliferative vitreoretinopathies characterized by EMT.
doi:10.1038/labinvest.2011.201
PMCID: PMC3961713  PMID: 22391957
ARPE-19 cells; contact inhibition; epithelial-mesenchymal transition (EMT); proliferation; Wnt signaling; Smad; ZEB
2.  Terpinen-4-ol is the Most Active Ingredient of Tea Tree Oil to Kill Demodex Mites 
Purpose
To determine the active ingredient in tea tree oil (TTO) responsible for its reported killing effect on Demodex mites, the most common ectoparasite found in the human skin extending to the eye.
Methods
Using a reported in vitro killing assay to measure the survival time of adult Demodex folliculorum up to 150 minutes, we have screened serial concentrations of 13 of the 15 known ingredients of TTO (ISO4730:2004) that were soluble in mineral oil and examined their synergistic relationships in killing mites. The most potent ingredient was then tested for its efficacy in killing Demodex in vivo.
Results
All ingredients exhibited a dose-dependent killing effect. Besides Terpinen-4-ol, the order of relative potency did not correlate with the order of relative abundance in TTO for the remaining 12 ingredients. Terpinen-4-ol was the most potent ingredient followed by α-Terpineol, 1,8-Cineole and Sabinene. Terpinen-4-ol, the most abundant ingredient in TTO, was more potent than TTO at equivalent concentrations and its killing effect was even observable at a mere concentration of 1%. Terpinen-4-ol exhibited a significant synergistic effect with Terpinolene, but an antagonistic effect with α-Terpineol in killing mites (both P < 0.05). In vivo, Terpinen-4-ol was shown to eradicate mites.
Conclusions
The above finding suggests that deployment of Terpinen-4-ol alone should enhance its potency in killing Demodex mites by reducing the adverse and antagonistic effects from other ingredients in TTO.
Translational Relevance
Terpinen-4-ol can be adopted in future formulations of acaricides to treat a number of ocular and cutaneous diseases caused by demodicosis.
doi:10.1167/tvst.2.7.2
PMCID: PMC3860352  PMID: 24349880
terpinen-4-ol; tea tree oil; demodex; acaricide; ocular drug delivery
3.  Optimization of Ex Vivo Expansion of Limbal Epithelial Progenitors by Maintaining Native Niche Cells on Denuded Amniotic Membrane 
Purpose
Transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on epithelially denuded amniotic membrane (dAM) in supplemented hormonal epithelial medium (SHEM) is an alternative solution for treating corneal blindness due to limbal stem cell (SC) deficiency. Because the phenotype of limbal niche cells (NCs) is preserved better in serum-free modified embryonic stem cell (ESC) medium (MESCM) than SHEM, we question whether the aforementioned expansion protocol can be further optimized by maintaining limbal NCs using MESCM.
Methods
Collagenase-isolated limbal clusters were cultured on dAM in SHEM or MESCM for 8 to 10 days. Epithelial outgrowth sheets removed by dispase were subjected to real-time quantitative polymerase chain reaction (qPCR) and immunostaining for expression of corneal epithelial markers (p63α, pax6, and K12) and NC markers (FLK-1, CD34, CD31, PDGFR-B, and α-SMA). A total of 1000 single cells were seeded on 6-well dish containing 3T3 feeder layers for 12 to 14 days before rhodamine B staining.
Results
Epithelial outgrowth in SHEM showed a significant loss of corneal SC and ESC markers when compared with freshly collagenase-isolated limbal clusters. Although the epithelial outgrowth was slower in MESCM, epithelial cell size was consistently smaller than that found in SHEM. Furthermore, MESCM maintained a significantly higher percentage of PCK−/ Vim+ cells and exhibited a significant upregulation of NC markers and corneal epithelial SC markers (K15, Bmi-1, and Msi-1) than SHEM. Furthermore, the number of purported holoclones was significantly promoted in MESCM than SHEM.
Conclusion
These data collectively suggest that MESCM can be used to replace SHEM to further promote expansion of LEPC by maintaining limbal native NCs.
Translational Relevance
Effective ex vivo expansion of limbal epithelial SC is a first and important step toward the success of treating corneal blindness caused by limbal stem cell deficiency and paves the way for future applications in regenerative medicine.
doi:10.1167/tvst.2.7.1
PMCID: PMC3823523  PMID: 24222891
cornea; ex vivo expansion; limbus; niche cells; stem cells; stem cell niche
4.  Mesenchymal Stem Cells Derived from Human Limbal Niche Cells 
Purpose.
We investigated whether human limbal niche cells generate mesenchymal stem cells.
Methods.
Limbal niche cells were isolated from the limbal stroma by collagenase alone or following dispase removal of the limbal epithelium (D/C), and cultured on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), or coated or three-dimensional Matrigel in embryonic stem cell medium with leukemia inhibitory factor and basic fibroblast growth factor. Expression of cell markers, colony-forming units-fibroblast, tri-lineage differentiation, and ability of supporting limbal epithelial stem/progenitor cells were compared to limbal residual stromal cells.
Results.
Stromal cells expressing angiogenesis markers were found perivascularly, subjacent to limbal basal epithelial cells, and in D/C and limbal residual stromal cells. When seeded in three-dimensional Matrigel, D/C but not limbal residual stromal cells yielded spheres of angiogenesis progenitors that stabilized vascular networks. Similar to collagenase-isolated cells, D/C cells could be expanded on coated Matrigel for more than 12 passages, yielding spindle cells expressing angiogenesis and mesenchymal stem cells markers, and possessing significantly higher colony-forming units-fibroblast and more efficient tri-lineage differentiation than D/C and limbal residual stromal cells expanded on plastic in DMEM with 10% FBS, of which both lost the pericyte phenotype while limbal residual stromal cells turned into myofibroblasts. Upon reunion with limbal epithelial stem/progenitor cells to form spheres, D/C cells expanded on coated Matrigel maintained higher expression of p63α and lower expression of cytokeratin 12 than those expanded on plastic in DMEM with 10% FBS, while spheres formed with human corneal fibroblasts expressed cytokeratin 12 without p63α.
Conclusions.
In the limbal stroma, cells subjacent to limbal basal epithelial cells serve as niche cells, and generate progenitors with angiogenesis and mesenchymal stem cells potentials. They might partake in angiogenesis and regeneration during corneal wound healing.
In the limbal stroma, niche cells subjacent to limbal basal epithelial cells generate progenitors with angiogenesis and MSC potentials. They might partake in angiogenesis and regeneration during corneal wound healing.
doi:10.1167/iovs.12-10300
PMCID: PMC3423309  PMID: 22836771
5.  Angiogenesis Potential of Human Limbal Stromal Niche Cells 
Purpose.
The perivascular localization of stem cell (SC) niches suggests the presence of a vascular niche. We aimed to determine the angiogenesis potential of limbal niche cells (NCs).
Methods.
Human limbal NCs were isolated and serially passaged on plastic or coated Matrigel in embryonic SC medium containing BFGF and leukemia inhibitory factor before being reseeded in 3D Matrigel. Expression of angiogenesis markers was assessed by RT-qPCR and immunofluorescence staining. Their angiogenesis potential was measured by differentiation into vascular endothelial cells and by supporting vascular tube network formed by human umbilical vein endothelial cells (HUVEC) on Matrigel. Their support of limbal epithelial progenitor cells (LEPC) was examined in sphere growth formed by reunion in 3D Matrigel.
Results.
On plastic, limbal NC could be cultured only up to four passages before turning into myofibroblasts. In contrast, on coated Matrigel, they could be expanded for up to 12 passages with upregulation of markers suggestive of angiogenesis progenitors when reseeded in 3D Matrigel because they could differentiate into vascular endothelial cells and pericytes stabilizing the tube network formed by HUVEC. Although both expanded limbal NCs and HUVEC rejoined with LEPC to form spheres to upregulate expression of ΔNp63α, CK15, and CEBPδ, the former but not the latter abolished expression of CK12 keratin.
Conclusions.
Human limbal NCs continuously expanded on the basement membrane differentiate into angiogenesis progenitors that prevent differentiation of LEPC/SCs. They may partake in formation of the vascular niche and contribute to angiogenesis during wound healing.
Human limbal stromal niche cells have the potential of differentiating into angiogenesis progenitors and preventing corneal epithelial differentiation, implying their partaking in formation of the vascular niche and contributing to angiogenesis during wound healing.
doi:10.1167/iovs.11-9414
PMCID: PMC3374622  PMID: 22538425
6.  PTX3 Controls Activation of Matrix Metalloproteinase 1 and Apoptosis in Conjunctivochalasis Fibroblasts 
Purpose.
Conjunctivochalasis (CCh) is an age-related inflammatory ocular surface disease manifesting redundant, loose conjunctiva folds. The pathogenic role of Pentraxin 3 (PTX3) in controlling upregulation of matrix metalloproteinase 1 (MMP-1) and MMP-3 in CCh remains undefined.
Methods.
Cytolocation of PTX3 and apoptosis were compared by immunostaining and terminal deoxyribonucleotidyl transferase-mediated FITC-linked dUTP nick-end DNA labeling (TUNEL) assay between normal and CCh specimens containing the conjunctiva and the Tenon. Second to third cultures of normal and CCh fibroblasts were treated with or without Aprotinin, Batimastat, or N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH), followed by transfection with or without PTX3 siRNA, and TNF-α or IL-1β. Cell lysates and culture media were collected to assess apoptosis measured by the Cell Death Detection ELISA and expression of PTX3, MMP-1, and MMP-3 transcripts and proteins by quantitative RT-PCR and Western blot, respectively.
Results.
PTX3 immunostaining was negative in normal specimens, but strongly positive in the subconjunctival stroma of CCh specimens. More apoptotic cells were found in CCh samples than in normal specimens. Expression of PTX3 transcripts and protein was not constitutive in resting normal fibroblasts but was in resting CCh fibroblasts and was upregulated by IL-1β in both cell lysates and culture media of both fibroblasts. PTX3 siRNA further upregulated MMP-1 and MMP-3 transcripts in resting normal fibroblasts, but synergistically with IL-1β upregulated the expression of MMP-1 and MMP-3 transcripts only in CCh fibroblasts, with activation of MMP-1 more so than MMP-3. PTX3 siRNA knockdown also promoted cell death characterized by apoptosis and necrosis, and such cell death could be rescued by inhibitors against serine proteinase, MMP1, or MMP3.
Conclusions.
Perturbation of PTX3 expression might partake in apoptosis and pathogenesis of CCh by upregulating expression of MMP-1 and MMP-3, and activation of MMP-1 and MMP-3.
This work represents a major advance in the field in conjunctivochalasis (CCh) to link the pathogenic role of PTX3 in controlling transcription and activation of MMP-1 and MMP-3 in the conjunctival tissue.
doi:10.1167/iovs.11-9103
PMCID: PMC3390005  PMID: 22511625
7.  TSG-6 Controls Transcription and Activation of Matrix Metalloproteinase 1 in Conjunctivochalasis 
The authors believe this study represents a major advance in the field in conjunctivochalasis to link the pathogenic role of TSG-6 in controlling transcription and activation of MMP-1 in the conjunctival tissue.
Purpose.
To investigate the role of anti-inflammatory TSG-6 in controlling MMP-1 and MMP-3, which have been shown to be upregulated in conjunctivochalasis (CCh).
Methods.
Immunostaining of TSG-6 was compared between normal and CCh conjunctiva and Tenon's capsule. Second cultures of normal and CCh fibroblasts were transfected with or without TSG-6 siRNA and then with or without the addition of TNF-α or IL-1β. Cell lysates and culture media were collected to assess apoptosis with the use of ELISA and the expression of TSG-6, MMP-1, and MMP-3 transcripts and proteins with the use of qRT-PCR and Western blot analysis, respectively.
Results.
TSG-6 expression was constitutive in the in vivo normal conjunctival epithelium. Significantly more TSG-6–positive cells than normal specimens were noted in CCh subconjunctival tissue and Tenon's capsule. TSG-6 was constitutively expressed intracellularly by both resting normal and CCh fibroblasts but was secreted extracellularly only by resting CCh fibroblasts. Intracellular and extracellular TSG-6 proteins were markedly upregulated by TNF-α or IL-1β in normal and CCh fibroblasts. Active MMP-1 was found in CCh fibroblasts intracellularly and extracellularly, whereas only proMMP-1 was found intracellularly in normal fibroblasts. Knockdown by TSG-6 siRNA upregulated more MMP-1 than MMP-3 transcripts in normal and CCh fibroblasts. TSG-6 siRNA led to extracellular MMP-1 expression by normal fibroblasts such as CCh fibroblasts. This activation of MMP-1 was further enhanced by IL-1β. Cell apoptosis was higher in CCh fibroblasts and further aggravated by TSG-6 siRNA knockdown.
Conclusions.
TSG-6 exerts an anti-inflammatory function by counteracting the transcription of MMP-1 and MMP-3 and the activation of MMP-1. Dysfunction of TSG-6 might play a role in the pathogenesis of CCh.
doi:10.1167/iovs.11-8738
PMCID: PMC3339910  PMID: 22297496
8.  Isolation and Expansion of Human Limbal Stromal Niche Cells 
Limbal stromal niche cells heterogeneously express embryonic stem cell markers. They were isolated and expanded on different Matrigel substrate by maintaining their original phenotype. Their phenotype is critical to the prevention of differentiation of limbal epithelial progenitors.
Purpose.
Limbal stromal niche cells heterogeneously express embryonic stem cell (SC) markers. This study was conducted to isolate and expand them and to prove that their phenotype is critical for supporting SCs.
Methods.
Human limbus was isolated by dispase or collagenase. Single cells were seeded on coated, 2D, or 3D Matrigel and were serially passaged in modified embryonic SC medium (MESCM), supplemented hormonal epithelial medium (SHEM), or Dulbecco's modified Eagle's medium plus 10% fetal bovine serum (DF) before they were seeded in 3D Matrigel. Sphere growth was achieved by mixing expanded single cells with dispase-isolated epithelial cells in 3D Matrigel. Expression of SC markers was analyzed by qRT-PCR, immunofluorescence staining, and Western blot; SC clonal growth was measured on 3T3 feeder layers.
Results.
Collagenase, but not dispase, isolated subjacent mesenchymal cells, of which the expression of Oct4, Sox2, Nanog, Rex1, SSEA4, N-cadherin, and CD34 was promoted in MESCM more than SHEM or DF. Reunion of PCK+ and Vim+ cells generated spheres in 3D Matrigel, but spindle cells emerged on 2D or coated Matrigel. Serial passages on coated Matrigel resulted in rapid expansion of spindle cells, of which the expression of ESC markers had declined but could be regained after reseeding in 3D Matrigel in MESCM but not in SHEM or DF. Resultant epithelial spheres mixed with spindle cells expanded in MESCM expressed more p63α, less CK12, and more holoclones than those mixed with spindle cells expanded in DF.
Conclusions.
Limbal stromal niche cells expressing SC markers can be isolated and expanded to prevent differentiation and maintain clonal growth of limbal epithelial progenitors.
doi:10.1167/iovs.11-8441
PMCID: PMC3292364  PMID: 22167096
17.  Selective Activation of p120ctn-Kaiso Signaling to Unlock Contact Inhibition of ARPE-19 Cells without Epithelial-Mesenchymal Transition 
PLoS ONE  2012;7(5):e36864.
Contact-inhibition ubiquitously exists in non-transformed cells and explains the poor regenerative capacity of in vivo human retinal pigment epithelial cells (RPE) during aging, injury and diseases. RPE injury or degeneration may unlock mitotic block mediated by contact inhibition but may also promote epithelial-mesenchymal transition (EMT) contributing to retinal blindness. Herein, we confirmed that EMT ensued in post-confluent ARPE-19 cells when contact inhibition was disrupted with EGTA followed by addition of EGF and FGF-2 because of activation of canonical Wnt and Smad/ZEB signaling. In contrast, knockdown of p120-catenin (p120) unlocked such mitotic block by activating p120/Kaiso, but not activating canonical Wnt and Smad/ZEB signaling, thus avoiding EMT. Nuclear BrdU labeling was correlated with nuclear release of Kaiso through p120 nuclear translocation, which was associated with activation of RhoA-ROCK signaling, destabilization of microtubules. Prolonged p120 siRNA knockdown followed by withdrawal further expanded RPE into more compact monolayers with a normal phenotype and a higher density. This new strategy based on selective activation of p120/Kaiso but not Wnt/β-catenin signaling obviates the need of using single cells and the risk of EMT, and may be deployed to engineer surgical grafts containing RPE and other tissues.
doi:10.1371/journal.pone.0036864
PMCID: PMC3348893  PMID: 22590627
18.  A New Isolation Method of Human Limbal Progenitor Cells by Maintaining Close Association with Their Niche Cells 
In human corneal epithelium, self-renewal and fate decision of stem cells are highly regulated in a niche microenvironment called palisades of Vogt in the limbus. Herein, we discovered that digestion with dispase, which cleaves off the basement membrane, did not remove the entire basal epithelial progenitor cells. In contrast, digestion with collagenase isolated on cluster consisting of not only entire epithelial progenitor cells but also their closely associated mesenchymal cells because of better preservation of some basement membrane matrix. Collagenase isolated more basal epithelial progenitor cells, which were p63α+ and small in the size (8 μm in diameter), and generated significantly more holoclones and meroclones on 3T3 fibroblast feeder layers than dispase. Further, collagenase isolated more small pan-cytokeratin−/p63α−/vimentin+ cells with the size as small as 5 μm in diameter and heterogeneously expressing vimentin, Oct4, Sox2, Nanog, Rex1, Nestin, N-cadherin, SSEA4, and CD34. Maintenance of close association between them led to clonal growth in a serum-free, low-calcium medium, whereas disruption of such association by trypsin/EDTA resulted in no clonal growth unless cocultured with 3T3 fibroblast feeder layers. Similarly, on epithelially denuded amniotic membrane, maintenance of such association led to consistent and robust epithelial outgrowth, which was also abolished by trypsin/EDTA. Epithelial outgrowth generated by collagenase-isolated clusters was significantly larger in diameter and its single cells yielded more holoclones on 3T3 fibroblast feeder layers than that from dispase-isolated sheets. This new isolation method can be used for exploring how limbal epithelial stem cells are regulated by their native niche cells.
doi:10.1089/ten.tec.2010.0609
PMCID: PMC3129703  PMID: 21175372
19.  Epidermal Differentiation and Loss of Clonal Growth Potential of Human Limbal Basal Epithelial Progenitor Cells during Intrastromal Invasion 
Ex vivo expansion of human limbal explant is hindered by intrastromal invasion of human limbal basal epithelial progenitor cells. The fate of these invaded progenitor cells conceivably dictates the success of the ex vivo expansion protocol.
Purpose.
Intrastromal invasion by limbal basal epithelial progenitor cells in explant cultures is associated with epithelial-mesenchymal transition. It remains unclear whether intrastromal invasion is contingent on culturing conditions and whether invaded cells retain their progenitor status and original lineage.
Methods.
Human limbal explants were cultured on various culture substrates, with or without air-lifting (AL), and subjected to hematoxylin and eosin staining and immunostaining to pan-cytokeratins, p63α, ΔNp63, Pax6, CK10, and CK12. Single cells obtained by trypsin/EDTA from dispase-isolated epithelial sheets from both the outgrowth and the surface epithelium, or by collagenase from the remaining stroma, were seeded on 3T3 feeder layers.
Results.
Intrastromal invasion was verified in all seven explant cultures by positive pan-cytokeratin staining. Immunofluorescence staining revealed that invaded epithelial cells were positive for p63α and ΔNp63, with or without nuclear staining of Pax6. Double immunostaining to CK10 and CK12 revealed that squamous metaplasia induced by AL was noted on the surface epithelium but not in intrastromally invaded epithelial cells. On 3T3 feeder layers, both the outgrowth and the surface epithelium yielded significant numbers of holoclones and meroclones positive to ΔNp63 but negative to CK10 and CK12. In contrast, intrastromally invaded epithelial cells generated only paraclones negative to ΔNp63 and CK12 but positive to CK10 regardless of culturing conditions.
Conclusions.
Intrastromal invasion by limbal basal epithelial progenitor cells is universal in all explant culture conditions, explaining why there is a gradual decline of outgrowth potential. Alteration of the limbal stromal niche leads invaded epithelial cells to adopt an epidermal fate.
doi:10.1167/iovs.10-6376
PMCID: PMC3175945  PMID: 21527382
20.  Stratified epithelial sheets engineered from a single adult murine corneal/limbal progenitor cell 
The limbal region of the adult cornea contains stem cells which are ultimately responsible for regeneration of the corneal epithelium during wound repair. However, primarily-isolated murine corneal/limbal epithelial cells rapidly senesce on plastic in a serum-free low [Ca2+] medium, suggesting only transit amplifying cells are promoted. We developed a novel expansion method by seeding at a low cell density (<500 cells/cm2) and prolonging each culture time beyond the lifespan of transit amplifying cells (4 weeks). Expanded cells were uniformly small, negative to K12 keratin, but positive for p63 nuclear staining, and could be subcultured beyond 100 passages. After limiting dilution, one clone (TKE2) was selected that exhibited single cell clonal expansion with a doubling time of 34.2 hrs, and had normal karyotyping, but no anchorage-independent growth. A single cell could be continually expanded to a confluent monolayer on denuded amniotic membrane and became stratified by exposing to the air-medium interface. The resultant stratified epithelium expressed K14 keratin, involucrin, connexin 43 and p63, but not K12 keratin or Pax 6. However, expression of K12 could be up-regulated by increasing extracellular calcium concentration and addition of foetal bovine serum (FBS) at P12, but less so at P85. Therefore, this murine limbal/corneal epithelium-derived progenitor cell line still retained the plasticity for adopting corneal lineage differentiation, could be useful for investigating limbal niche cues that may promote corneal epithelial fate decision.
doi:10.1111/j.1582-4934.2008.00297.x
PMCID: PMC3225011  PMID: 18318692
cornea; epithelium; stem cell; regenerative medicine; culture; senescence and growth
21.  On the influence of neutrophils in corneas with necrotizing HSV-1 keratitis following amniotic membrane transplantation☆ 
Experimental eye research  2007;85(3):335-345.
Necrotizing herpetic stromal keratitis (HSK) in mice rapidly improved after amniotic membrane transplantation (AMT). In this study we determined the fate of polymorphonuclear neutrophils (PMN) after AMT. AMT or tarsorrhaphy (T) was performed in BALB/c mice with ulcerative HSK. After 2 days, corneas were studied histologically and by transmission electron microscopy (TEM). CD11b, Gr-1, and TUNEL-positive cells were identified. Macrophages were depleted by subconjunctival injection of dichloromethylene-diphosphonate-liposomes (Cl2MDP-LIP) before AMT. Corneas were studied for interleukin (IL)-1α, IL-2, interferon (IFN)-γ, CXCL1, CXCL2, and tumor necrosis factor (TNF)-α production by ELISA. PMN-enriched cell preparations co-cultured with amniotic membrane (AM) or with AM and such recombinant (r) cytokines as rIL-1α, rIL-2, and rTNF-α or supernatants from activated lymphocytes were investigated by flow cytometry (Annexin-V/7-AAD and TUNEL), and a dimethylthiazolyl-diphenyltetrazolium-bromide (MTT)-viability assay. Corneas in the AMT mice had less inflammation, fewer PMN-like cells and fewer CD11b+, and Gr-1+ cells (P < 0.01), but a higher ratio of apoptotic to viable PMN-resembling cells (P < 0.01) than the T mice. Phagocytic removal of apoptotic PMN-like cells by macrophages was evident in the AMT group. After Cl2MDP-LIP treatment, the corneas had more cell debris and apoptotic cells with PMN-like morphology. The concentrations of IL-1 α, IL-2, CXCL1, and TNF-α were reduced in corneas of the AMT group as compared to that of the T group, while the concentration of CXCL2 was increased. Apoptosis of PMN-resembling cells was detected following cocultivation with AM, even when proinflammatory cytokines were present. Resolution of corneal inflammation in mice with necrotizing HSK after AMT is associated with increased apoptosis of PMN-like cells, reduction of pro-inflammatory cytokines, an increase of CXCL2, and increased removal of apoptotic PMN-like cells by macrophages.
doi:10.1016/j.exer.2007.05.009
PMCID: PMC3209667  PMID: 17637463
amniotic membrane transplantation; herpetic stromal keratitis; apoptosis; neutrophils; macrophage; macrophage depletion; infection; cytokines
22.  A Novel Method of Isolation, Preservation, and Expansion of Human Corneal Endothelial Cells 
Purpose
To explore new strategies for effective isolation, preservation, and expansion of human corneal endothelial cells (HCECs).
Methods
Human corneal Descemet’s membrane and corneal endothelial cells were digested with collagenase A or Dispase II in supplemented hormonal epithelial medium (SHEM) for 1.5 to 16 hours. HCEC aggregates derived from collagenase A digestion were preserved in serum-free medium with low or high calcium for up to 3 weeks. Cryosections of HCEC aggregates were subjected to immunostaining with ZO-1, connexin 43, type IV collagen, laminin-5, and perlecan, and apoptosis was determined by TUNEL or cell-viability assay. For expansion, HCEC aggregates were seeded directly or after brief treatment with trypsin/EDTA in SHEM, with or without additional bovine pituitary extract (BPE), nerve growth factor (NGF), or basic fibroblast growth factor (bFGF). The resultant HCECs were immunostained with ZO-1, connexin 43, and Ki67.
Results
Digestion with collagenase A, but not Dispase, of the stripped Descemet’s membrane generated HCEC aggregates, which preserved cell–cell junctions and basement membrane components. High cell viability of HCEC aggregates was preservable in a serum-free, high-calcium, but not low-calcium, medium for at least 3 weeks. Brief treatment of HCEC aggregates with trypsin/EDTA resulted in a higher proliferation rate than without, when cultured in SHEM, and the resultant confluent monolayer of hexagonal cells retained cell–cell junctions. However, additional BPE, NGF, or bFGF did not increase cell proliferation, whereas additional BPE or bFGF disrupted cell–cell junctions.
Conclusions
Collagenase A digestion successfully harvested aggregates with viable HCECs that were preservable for at least 3 weeks in a serum-free, high-calcium medium and, with brief trypsin/EDTA treatment, expanded in the SHEM into a monolayer with hexagonal cells that exhibited characteristic cell junctions.
doi:10.1167/iovs.06-1126
PMCID: PMC3196988  PMID: 17251457
23.  Human Amniotic Epithelial Cells as Novel Feeder Layers for Promoting Ex Vivo Expansion of Limbal Epithelial Progenitor Cells 
Stem cells (Dayton, Ohio)  2007;25(8):1995-2005.
Human amniotic epithelial cells (HAECs) are a unique embryonic cell source that potentially can be used as feeder layers for expanding different types of stem cells. In vivo, HAECs uniformly expressed pan-cytokeratins (pan-CK) and heterogeneously expressed vimentin (Vim). The two phenotypes expressing either pan-CK (+)/Vim(+) or pan-CK(+)/Vim(−) were maintained in serum-free media with high calcium. In contrast, all HAECs became pan-CK (+)/Vim(+) in serum-containing media, which also promoted HAEC proliferation for at least 8 passages especially supplemented with EGF and insulin. Mitomycin C-arrested HAEC feeder layers were more effective in promoting clonal growth of human limbal epithelial progenitors than conventional 3T3 murine feeder layers. Cells in HAEC-supported clones were uniformly smaller, sustained more proliferation, and expressed less CK12 and connexin 43, but higher levels of stem cell-associated markers such as p63, Musashi-1 and ABCG2 than those of 3T3-supported clones. Subculturing of clonally expanded limbal progenitors from HAEC feeder layers, but not from 3T3 feeder layers, gave rise to uniformly p63-positive epithelial progenitor cells as well as nestin-positive neuronal-like progenitors. Collectively, these results indicated that HAECs can be used as a human feeder layer equivalent for more effective ex vivo expansion of adult epithelial stem cells from the human limbus.
doi:10.1634/stemcells.2006-0677
PMCID: PMC3197019  PMID: 17495107
ABCG2; amniotic membrane; amniotic epithelial cells; ex vivo expansion; feeder layers; connexin 43 cytokeratins; K12; limbus; Musashi-1; neuronal progenitors; plasticity; progenitor cells; p63; stem cells
24.  The Fate of Limbal Epithelial Progenitor Cells during Explant Culture on Intact Amniotic Membrane 
Purpose
The clinical success of treating corneas with total limbal stem cell deficiency using limbal biopsy explants cultured on intact amniotic membrane (iAM) relies on ex vivo expansion of limbal epithelial progenitor cells. However, the ultimate fate of limbal epithelial progenitor cells in the explant remains unclear.
Methods
Human limbal explants were cultured on iAM for 2 weeks and then removed and transferred to a new iAM until passage 3. The outgrowth surface area of each passage was measured and compared. For each passage, clonogenicity on 3T3 fibroblasts feeder layers was compared among progenitor cells removed from the outgrowth, the explant surface, and the remaining stroma. Cryosections of the explant and the outgrowth were detected with p63, vimentin, pancytokeratin, and the basement membrane components type VII and IV collagen and laminin 5 antibodies.
Results
The outgrowth surface area significantly decreased from passage (P)1 to P3. The total number of epithelial cells that were isolated from the explant surface also decreased from before culture (P0) to P1, became stable from P1 to P2, but was uncountable at P3. Clonogenicity significantly declined from P1 to P3 for the epithelium derived from the explant surface and the outgrowth epithelium; the extent was less in the former than in the latter at P2 and P3. In addition, groups of epithelial cells invaded the limbal stroma of the explants from P1 to P3; p63(+)/pancytokeratin(−) and p63(+)/vimentin(+) cells also presented in the limbal stroma. Increasing fibroblast, but not epithelial, colonies were observed from cells isolated from the remaining limbal stroma when seeded on 3T3 fibroblast feeder layers from P1 to P3.
Conclusions
During ex vivo expansion on iAM, some limbal epithelial progenitor cells indeed migrate onto iAM from the explant surface, whereas some also invade the limbal stroma, very likely undergoing epithelial-mesenchymal transition. This new information should be taken into account in formulating new strategies to improve the expansion protocol.
doi:10.1167/iovs.06-0514
PMCID: PMC3197022  PMID: 17251456
25.  Amniotic membrane induces apoptosis of interferon-γ activated macrophages in vitro 
Experimental eye research  2005;82(2):282-292.
Amniotic membrane (AM) used as a temporary or permanent graft for ocular surface reconstruction has a potent anti-inflammatory effect. We would like to investigate the mechanism whereby AM induces macrophage apoptosis in vitro. Mouse macrophages, Raw 264.7 cells, were cultured on plastic, type I collagen, corneal stromal slice or AM stromal matrix in serum-free medium with or without interferon-γ (IFN-γ). Cells were stained by LIVE/DEAD assay, Hoechst-33342, and TUNEL assay for cell death and apoptosis. Cell lysates and conditioned media were analysed by Cell Death Detection ELISA assay for quantitation of apoptosis. Conditioned media were also analysed by Griess assay for the nitrite concentration and ELISA assay for tumour necrosis factor alpha (IFN-α) concentration. Lysates of cells were subjected to Western blot analyses of IKK-α, IKK-β, p65 (RelA) subunit of nuclear factor κB (NF-κB), total Akt, phospho-Akt (Ser473), and phospho-FKHR (Thr24)/phosphor-FKHRL1 (Thr32). At 48 hr after cultivation, cells showed a low level of apoptosis when cultured on plastic, type I collagen and corneal stromal slice with or without IFN-γ and on AM without IFN-γ. Nevertheless, cells showed a significant increase of apoptosis when cultured on AM with IFN-γ activation, and this phenomenon became apparent only after 48 hr. IFN-γ-activated macrophages on plastic continuously produced nitric oxide (NO) and IFN-α during 72 hr culturing. In contrast, there was no NO and IFN-α production after 48 hr culture on AM. NO inhibitors, L-NMMA and L-NIL, attenuated NO production of IFN-γ-activated macrophages on AM, while apoptosis was not decreased accordingly. Expression of IKK-α, IKK-β, p65 (RelA) subunit of NF-κB total Akt, phosopho-Akt (Ser473), and phospho-FKHR (Thr24)/FKHRL1 (Thr32) was all down-regulated in IFN-γ-activated macrophages cultured on AM. In conclusion, AM stromal matrix induces apoptosis of IFN-γ activated, but not non-activated macrophages, not through the generation of NO, but instead by down-regulating anti-apoptotic NF-κB and Akt-FKHR signalling pathways.
doi:10.1016/j.exer.2005.06.022
PMCID: PMC3193177  PMID: 16109408
amniotic membrane; apoptosis; interferon-γ; macrophages

Results 1-25 (47)