Human Limbal Cells Research Articles

Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC) and epithelial limbal (LSC) stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8), E8 supplemented with EGF (E8+) or Green’s medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green’s media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

Regulation of Human Adenovirus Replication by RNA Interference

Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy.

Mechanism of embryonic stem cells microenvironment enhancing stemness and inhibiting apoptosis of human limbal stem cells

Background The fate of adult stem cells is associated with its surrounding microenviroment.Our previous work found that embryonic stem cells (ESCs) micro-environment enhance the stemness of human limbal stem cells (LSCs), but its mechanism has not been elucidated. Objective This study was to explore the molecular mechanism of ESC micro-environment enhancing the stemness and inhibiting the apoptosis of LSCs. Methods Human LSCs were cultured by explant culture method with CnT-20 medium and CnT-20+ 20%ES culture supernatant (ESC-CM), respectively.Colony formation assay was used to analyze the proliferation ability of cells.Telomerase reverse transcriptase (TERT) siRNA(19-25nt siRNA) or siRNA(sc-37007) was transfected into the cells of ESC-CM group.Apoptosis and mitochondrial membrane potential were assayed by flow cytometry, and the expressions of telomerase and reactive oxygen species (ROS) in TERT siRNA- or siRNA-F-transfected cells by immunofluorescence and flow cytomery.RT-PCR, immunofluorescence staining and Western blot were employed to determine the expressions of p63, ATP-binding cassette transporer G2 (ABCG2), integrin β1 mRNA and proteins and cytokeratin 3(CK3) in the cells.The levels of focal adhesion kinase (FAK), Akt, glycogen synthase kinase 3β (GSK3β) and p21 protein and phosphorylation proteins in the cells were detected by Western blot. Results The LSCs presented an increased proliferative capacity and passaged to the eighth generation with the colony-forming efficiency (CFE) of (7.6±0.6)% in ESC-CM group, but the cells to the sixth generation with the CFE of (5.6±0.6)%, showing a significant difference between them(t=4.454, P=0.011). The apoptotic rates of the cells from 2 through 6 generations were lower in the ESC-CM group than those in the CnT-20 group (all at P 0.05), but significantly declined expressions of CK3 mRNA and protein were found in the ESC-CM group compared with the CnT-20 group (all at P<0.01). However, the expressions of p63, ABCG2, integrin β1 mRNA and proteins and TERT protein in the second generation of the cells were significantly higher in the ESC-CM group compared with the CnT-20 group (all at P<0.01). The telomerase activity was (4.83±0.67) % in the siRNA-TERT transfected group, which was significantly lower than (46.71±1.22) % of the siRNA-F transfected group (t=52.116, P=0.000). The expression of pFAK, pAkt, pGSK3β proteins were weakened, but the expression of p21 was increased in the ESC-CM group after addition of FAK inhibitor, GSK3β inhibitor and TERT-siRNA transfected group.Mitochondrial membrane potential in the second generation of cells was elevated in the ESC-CM group in comparison with the CnT-20 group and the siRNA-TERT transfected group (all at P<0.01), and the rates of ROS positively reaction was lower in the ESC-CM group and the siRNA-F transfected group than those of the CnT-20 group and siRNA-TERT transfected group (all at P<0.01). Conclusions ESC-CM culture system can effectively keep the stemness of LSCs and inhibit apoptosis.ESC-CM culture system plays functions probably via telomerase-p21-mitochondrial axis and the activation of the FAK/Wnt signaling pathways. Key words: Embryonic stem cells; Niche; Telomerase; Limbal stem cells; Signaling pathway; Apoptosis; Colony-forming units

Effects of amniotic membrane extract on primary human corneal epithelial and limbal cells.

To assess the effects of amniotic membrane extract (AMX) on cellular activity of primary human corneal epithelial (HCE) cells under mechanical and oxidative stress, and on human limbal cells under oxidative stress. Corneal mechanical stress was simulated with a linear scratch in confluent HCE cell plates, then incubated with 0.1% AMX for 48 and 72 h. Subjecting HCE cultures to 0.5 mmol/L tertiary-butylhydroperoxide for 1 h simulated an oxidative stress. 0.1% AMX-treated cultures were compared with controls at 24 and 48 h using cellular viability assay, along with 12-h AMX pretreatment and human limbal cell comparisons. Mechanical stress on HCE cultures revealed a statistically significant distance ratio at 48 and 72 h in favour of 0.1% AMX-treated cultures (P = 0.021 and 0.035, respectively). Oxidative stress did not reveal any significant difference in cellular viability of AMX-treated versus control cultures. Twelve hour AMX pre-treatment prior to oxidative stress revealed a significant difference after 24 h from oxidative injury (73.3% AMX vs. 66.0% control, P = 0.035), but not after 48 h. Human limbal cells demonstrated significantly improved oxidative viability compared with HCE cells, with (91.0% vs. 82.0% control, P = 0.017) and without 0.1% AMX pre-treatment (91.2% vs. 83.7% control, P = 0.019). HCE cells treated with AMX healed faster after mechanical insult, suggesting a potential benefit in acute corneal injuries. Under oxidative stress, human limbal cells, a more proliferative cell type, showed superior viability compared with HCE cells.

Application of fetal limbal stem cell in the treatment of pseudopterygium

Objective To study the clinical effect of fetal limbal stem cell in the treatment of pseudopterygium.Methods 16 eyes of 16 patients had been peformed the operation of pseudopterygium excision combined with fetal limbal stem cell transplantation.Fresh human fetal limbal stem cell with donors' gestational age of 28 weeks or more were used intraoperatively.The medicine preventing immune rejection were used after operation.Corneo-conjunctival healing,immune rejection,palindromia of pterygium and intraocular pressure were observed in the follow-up.Results The patients were followed up for (9.4 ± 2.4)months.The average time for the corneal epithelial healing was (3.8 ± 1.3) d.In the follow-up,13 cases (81.3 ％) of corneal epithelial continued complete,1 case (6.3 ％) recurred pseudoperygium.The number of epithelial rejection were 7 cases,3 cases,0 case and 0 case at 1 m,3 ms,6 ms and at the last follow-up time after operation,but the rejection was controlled through medication,the average time of medicine treatment was (3.9 ± 1.3) d.At 1,3,and 6 months after surgery and the last follow-up,the mean rating value of graft rejection were 0.6 ± 0.9,1.0 ± 1.1,0.3 ± 0.8,0.3 ± 0.8.Six months after operation,there were 3 cases of graft dissolution.Two cases were significantly improved by anti-rejection medication,another one had continued dissolution and the graft failed to survive.Conclusion Preliminary results are satisfactory for the treatment of pseudopterygium caused by physic and chemical damage through transplanting fetal limbal stem cell,it provides a new idea for the clinical treatment of those diseases. Key words: Fetal; Stem cell, limbal; Tissue transplantation; Pseudopterygium

The impact of the bone marrow-derived mesenchymal stem cell on the microenvironment of the human limbal stem cell

Background When limbal stem cell deficiency (LSCD) occurs,not only the limbal stem cells (LSCs) were damaged,but also the LSCs matrix microenvironment was under destruction.The treatment of LSCD include both replenishing of stem cells and restoration of microenvironment.So far,the method to improve the microenvironment of LSCD still exist limitation and urgently need to establish more appropriate microenvironment for the LSCs growth in vitro.Objective This study was to investigate whether the human bone marrow-derived mesenchymal stem cells (BMSCs) can be used as the ideal cells to repair limbal microenvironment and its possible mechanism of repairing limbal microenvironment during the human LSCs amplification in vitro as feeder cells.Methods BMSCs were cultured and passaged in vitro,and flow cytometry was used to assay the expressions of CD45,CD71,CD90,CD105 and HLA-DR and directionally induced BMSCs to the osteoblasts and adipocytes.BMSCs were treated using mitomycin C (MMC) to use as the feeder cells.LSCs were separately co-cultured with BMSCs,Swiss-3T3 feeder cells and free-feeder cells,and colony-forming efficiency (CFE) of the LSCs was compared among different co-cultured groups.LSCs were then cultured sequentially and identified by flow cytometry.Expression of cytokines in BMSCs was confirmed by reverse transcriptional polymerase chain reaction (RT-PCR).Results Cultured BMSCs showed a good homogeneity,with a lot of expressions of interstitial cell markers such as CD71,CD90,CD105 and less expressions of hematopoietic cell markers including CD45 and HLA-DR.After separately cocultured with feeder cells for 12 days,the CFE of the LSCs co-cultured with BMSCs,Swiss-3T3 and no feeder cells was 3.67％ ±0.58％,4.30％ ± 1.54％ and 0.20％ ±0.10％,showing a statistical significant difference among the three groups(F =15.420,P =0.040).There was no statistically significant difference in the C FE of the LSCs between the BMSCs feeder group and the Swiss-3T3 feeder cells group(P =0.456),between the BMSCs feeder group and the free-feeder cells group or the Swiss-3T3 co-culture group and the free-feeder cells group (P =0.005,0.002).LSCs presented with a positive response for ABCG2 antigen in the co-cultured with BMSCs group.Basic fibroblast growth factor(bFGF),stem cell factor (SCF) and N-cadherin(N-cad) were positively expressed in the BMSCs as feeder cells.Conclusions Human BMSCs-derived feeder cells can improve the growth of the stromal microenvironment of the LSCs and enhance their proliferation ability.Human BMSCs are suitable for engineering of epithelial sheets. Key words: Bone marrow-derived mesenchymal stem cell; Limbal stem cell; Feeder cell; Cell culture in vitro

Laser scanning confocal microscopy-assisted obtain of limbal tissue for the ex vivo culture of human limbal epithelial stem cells and identification

Background Human limbal allograft transplantation or limbal autograft transplantation are the primary approaches to the severe corneal-blindness,but their application in clinic were limited because of the defects of donor material.With the development of tissue engineering technology,transplantation of in vitro cultured limbal epithelial stem cells is being an advanced management.Objective The aim of this work was to expand human limbal epithelial stem cells ex vivo under the guidance of confocal microscope and to lay the foundation for fabricating ex vivo cultured cell sheets.Methods Ten eyes of ten patients were examined with the Heidelberg Retina Tomography Ⅲ Rostock Cornea Module(HRT3-RCM)to elucidate the structure of the human corneoscleral limbus and to correlate limbal epithelial dimensions.According to the analysis of the images of limbal epithelia,the limbal tissues provided by Eye Bank of Henan Eye Institute were cut into suitable explants.Then,this study was conducted to expand limbal epithelial stem cells ex vivo on denuded amniotic membrane.The phenotypes of primary cultured cells were evaluated by morphology and immunofluorescent staining with antibodies for limbal epithelial stem cell markers (p63,cytokeratinl9)and differentiation markers(keratin 3,involucrin).This experimental procedure was approved by the Ethic Committee of Henan Provincial People's Hospital.The written informed consent was obtained from subjects before initiation of any examination.Results The palisade morphology of human limbus was imaged clearly on the laser scanning in vivo confocal microscopy and many hyperreflective cells were observed in palisade basal cells.The cell-island phenomenon was seen in the basement membrane under the laser scanning in vivo confocal microscopy.The oblique sections of limbus showed many papilla-like epithelial columns below the superficial limbal epithelia.Throughout the experiment duration,the epithelial cells grew well with the migration rates from limbal tissue (68.62± 16.94)％ and the migration time(5.83 ±2.04)days,which depended on the tissue freshness.Compared with the second and forth batch of tissue,the migration rates of the third and sixth batch of tissues were significantly higher(P＜0.05),and the migration time was evidently longer in the forth and sixth batch of tissue compared with the first,second,third and fifth batch(P＜0.05).The positively expressing rates in the cultured corneal stem cells were 4.05％ and 36.52％ for p63,26.07％ and 40.55％ for CK19,57.88％ and 40.81％ for K3,64.66％ and 59.19％ for involucrin.Conclusion Human limbal epithelial stem cells can be successfully and purposefully obtained from the limbal tissue based on the guidance confocal miscroscope.The cultured corneal stem cells can grow well on the denuded amniotic membrane Key words: Cornea/limbus ; stem cells ; Laser scanning confocal microscopy; Cell culture; Markers

Corneal surface reconstruction - a short review

Corneal surface reconstruction - a short review

Establishment of a corneal epithelial cell line spontaneously derived from human limbal cells

The objective of this study was to establish a spontaneously derived human corneal epithelial cell line from a normal human limbus that retains differentiation potential and proliferative properties under continuous cell culture. After 50 passages of epithelial cells obtained from human limbal tissue a cell line spontaneously emerged. The immortalized cells showed a cobblestone appearance and displayed dense microvilli on their apical cell surface membrane. Colony forming efficiency was 5–6% and population doubling time was 19.6 h. In the mRNA level, cytokeratin (CK) 3 and 12 were detected in this cell line. In the protein level, the cells expressed CK3, CK12, CK14, CK19, vimentin, and some other proteins such as F-actin and β-tubulin and β 1-integrin. They lacked p63. The immortalized cells had a heteroploid karyotype, but did not exhibit tumorigenic features. When cultured on an air–liquid interface the cells could form stratified multilayer epithelia. In summary, all these results indicated that a new human corneal epithelial cell line was spontaneously established from normal limbal tissue through serial culture. This cell line would be useful for studies of corneal epithelial biology and reconstructive corneal tissue engineering.