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 [Ca(2+)] medium, suggesting only transit amplifying cells are promoted. We developed a novel expansion method by seeding at a low cell density (<500 cells/cm(2)) 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 lim-bal/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.     

Telomerase activity is sufficient to bypass replicative senescence in human limbal and conjunctival but not corneal keratinocytes

The human ocular surface is covered by the conjunctival, corneal and limbal stratified epithelia. While conjunctival stem cells are distributed in bulbar and forniceal conjunctiva, corneal stem cells are segregated in the basal layer of the limbus, which is the transitional zone between the cornea and the bulbar conjunctiva. Keratinocyte stem and transient amplifying (TA) cells when isolated in culture give rise to holoclones and paraclones, respectively. Keratinocyte replicative senescence ensues when all holoclones have generated paraclones which express high levels of p16(INK4a). In the present study, we show that enforced telomerase activity induces the bypass of replicative senescence in limbal and conjunctival keratinocytes, without the inactivation of the p16(INK4a)/Rb pathway or the abrogation of p53 expression. hTERT-transduced limbal and conjunctival keratinocytes are capable to respond to both growth inhibitory and differentiation stimuli, since they undergo growth arrest in response to phorbol esters, and activate p53 upon DNA damage. Following a sustained PKC stimulation, occasional clones of p16(INK4a)-negative cells emerge and resume ability to proliferate. Telomerase activity, however, is unable to induce the bypass of senescence in corneal TA keratinocytes cultured under the same conditions. These data support the notion that telomere-dependent replicative senescence is a general property of all human somatic cells, including keratinocytes, and suggest that telomerase activity is sufficient to extend the lifespan only of keratinocytes endowed with high proliferative potentials (which include stem cells), but not of TA keratinocytes.     

Isoforms of p63 in corneal stem cells cultured on human amniotic membrane

The p63 gene supports stem cell proliferation and regulation in epithelial cells. In this study, corneal epithelial cells were cultured on human amniotic membrane (HAM) and investigated for p63 and its isoform genes. Human limbal biopsies obtained from cadaveric donor eyes were cultivated on intact and denuded HAM. Transactivation (TA) specific domain was positive in the limbal cells cultured over denuded HAM and negative on others. TAp63α,β,γ isoforms are negative in all the limbal cells cultured on intact, denuded and limbal tissues but not in cornel epithelial tissue. p63α isoform is present in all except on denuded HAM. αβ sharing region is not expressed only in cornel epithelial tissue. γ isoform is expressed in all the samples. ΔNp63α region is present in cells cultured over the intact HAM whereas it is negative on the cells cultured over the denuded HAM. The other isoforms such as ΔNp63β and ΔNp63γ are negative in all samples. The limbal cells cultured over the intact HAM were able to maintain high proliferative potential when compared to denuded HAM. Thus, p63 isoforms plays a biological function to retain the proliferative capacity of corneal epithelial cells and maintains the stemness when cultured on intact HAM.     

小鼠角膜上皮祖细胞系TKE2的表型研究

目的：观察小鼠角膜上皮祖细胞系TKE2在扩增以及分化状态下的角蛋白及干细胞标志物的表达情况。方法小鼠角膜上皮祖细胞系TKE2在无血清培养基Keratinocyte-SFM （KSFM）以及含10﹪胎牛血清（FBS）的DMEM培养基中培养,约70﹪融合时进行角蛋白10、12、14、15、16（K10、K12、K14、K15、K16）以及Connexin43、ABCG2的免疫荧光染色,以及Ki67、P63、PCNA的免疫细胞化学染色。结果无血清培养状态下的TKE2细胞呈克隆样生长,克隆内所有细胞呈ABCG2、K14、Ki67、PCNA以及P63阳性,K15阳性细胞散在分布,K16阳性细胞呈片状分布于克隆中央区,K10、K12以及Connexin43染色为阴性。在含有10﹪胎牛血清的DMEM中培养2 d后,细胞明显增大, ABCG2、K15、P63、Ki67以及PCNA转为阴性,克隆内只有少量细胞呈K16、K14阳性染色, K10、K12、Connexin43仍为阴性。结论 TKE2细胞具有角膜上皮干细胞特性,可以作为角膜缘上皮干细胞表型维持和分化诱导研究的良好工具。     

Basement membrane heterogeneity and variation in corneal epithelial differentiation

We have previously shown that the expression of a major 64-Kda keratin (K3) in corneal epithelium is site-related. It is found suprabasally in limbal epithelium, but uniformly (basal cells included) in central corneal epithelium. In the present study, we used a panel of antibodies against various components of corneal epithelial basement membrane to investigate a possible correlation between basement membrane heterogeneity and differential (basal vs. suprabasal) K3 keratin expression. One of these antibodies, AE27, stains human conjunctival basement membrane weakly, limbal basement membrane heterogeneously, and central corneal basement membrane strongly. Basal cells resting on basement membrane that stains strongly with AE27 tend to stain with monoclonal antibody AE5, which recognizes keratin K3. Basal cells on basement membrane staining weakly with AE27 tend not to stain with AE5. No such correlation exists between AE5 staining and type IV collagen, which is detectable immunohistochemically in conjunctival and limbal basement membrane, but not in corneal basement membrane overlying Bowman's layer. These results suggest that basement membrane of human corneal/conjunctival epithelium can be divided into at least three domains: the conjunctival basement membrane (type IV collagen-positive, AE27-weak), the limbal basement membrane (type IV collagen-positive, AE27-strong), and corneal basement membrane (type IV collagen-negative, AE27-strong). The results also raise the possibility that basement membrane heterogeneity may play a functional role in regulating keratin expression and other aspects of differentiation of corneal epithelium; more experiments are needed to test this hypothesis.     

Cultivation and differentiation characteristics of human limbal progenitor cells

BACKGROUND: The aim of this study was to investigate whether limbal progenitor cells can be cultured, expanded and differentiated in vitro not only to enter corneal differentiation but also towards RPE (retinal pigment epithelium) characteristics. METHODS: A 3mm broad strip of human corneoscleral limbal tissue was digested enzymatically and cells were set into cell culture. Differentiation status and characteristics, proliferation and phagocytotic activity were assessed by immunocytochemical staining in combination with digital and confocal microscopy. RESULTS: Immunocytological analysis revealed expression of Nestin and p63 marker suggesting progenitor cell properties. Mitotic activity was demonstrated by BrdU (bromodesoxyuridine) uptake. Upon consecutive passages, corneal differentiation markers were predominantly expressed. Phagocytotic activity was demonstrated via uptake of FITC (fluorescein isothiocyanate) labelled latex beads. RPE markers Bestrophin and Cytokeratin 8/18 as well as glial marker GFAP and neuronal marker MAP with respective controls were negative indicating no differentiation towards characteristics of retinal pigment epithelium or neural and glial lineage. CONCLUSIONS: The results suggest that isolation and cultivation of proliferating and phagocytotic cells from the human corneal limbus was achieved which showed characteristics of both progenitor and differentiated corneal cells. No evidence was found for the hypothesis of spontaneous differentiation potential towards RPE lineage or neuronal characteristics, providing evidence of the inherent directional capacity of limbal progenitor cells.     

Investigation of K14/K5 as a stem cell marker in the limbal region of the bovine cornea

Background

Identification of stem cells from a corneal epithelial cell population by specific molecular markers has been investigated previously. Expressions of P63, ABCG2 and K14/K5 have all been linked to mammalian corneal epithelial stem cells. Here we report on the limitations of K14/K5 as a limbal stem cell marker.

Methodology/Principal Findings

K14/K5 expression was measured by immunohistochemistry, Western blotting and Real time PCR and compared between bovine epithelial cells in the limbus and central cornea. A functional study was also included to investigate changes in K5/14 expression within cultured limbal epithelial cells undergoing forced differentiation. K14 expression (or its partner K5) was detected in quiescent epithelial cells from both the limbal area and central cornea. K14 was localized predominantly to basal epithelial cells in the limbus and suprabasal epithelial cells in the central cornea. Western blotting revealed K14 expression in both limbus and central cornea (higher levels in the limbus). Similarly, quantitative real time PCR found K5, partner to K14, to be expressed in both the central cornea and limbus. Following forced differentiation in culture the limbal epithelial cells revealed an increase in K5/14 gene/protein expression levels in concert with a predictable rise in a known differentiation marker.

Conclusions/Significance

K14 and its partner K5 are limited not only to the limbus but also to the central bovine cornea epithelial cells suggesting K14/K5 is not limbal specific in situ. Furthermore K14/K5 expression levels were not lowered (in fact they increased) within a limbal epithelial cell culture undergoing forced differentiation suggesting K14/K5 is an unreliable maker for undifferentiated cells ex vivo.     

Rat limbal epithelial side population cells exhibit a distinct expression of stem cell markers that are lacking in side population cells from the central cornea

The side population (SP) phenotype is shared by stem cells in various tissues and species. Here we demonstrate SP cells with Hoechst dye efflux were surprisingly collected from the epithelia of both the rat limbus and central cornea, unlike in human and rabbit eyes. Our results show that rat limbal SP cells have a significantly higher expression of the stem cell markers ABCG2, nestin, and notch 1, compared to central corneal SP cells. Immunohistochemistry also revealed that ABCG2 and the epithelial stem/progenitor cell marker p63 were expressed only in basal limbal epithelial cells. These results demonstrate that ABCG2 expression is closely linked to the stem cell phenotype of SP cells.     

Oxygen tension affects terminal differentiation of corneal limbal epithelial cells

Oxygen concentration has been shown to be crucial in the proliferation and differentiation of various types of cells, while the impact of oxygen tension on the lineage commitment of epithelial cells remains elusive. In this study, we investigated the effect of hypoxia on the differentiation of corneal limbal epithelium using an ex vivo squamous metaplasia model. Under normoxic conditions when exposed to air, the hyperproliferation and abnormal epidermal-like differentiation of human corneal limbal epithelium was induced, whereas when exposed to air under hypoxic conditions, although we observed augmented proliferation, the abnormal differentiation was inhibited. The Notch signaling pathway was activated in hypoxic cultures, whereas the p38 MAPK signaling pathway was downregulated. The addition of Notch inhibitor under hypoxic conditions restored the activation of p38 MAPK and resulted in the recidivation of limbal epithelial cells to epidermal-like differentiation. Moreover, the epidermal-like differentiation of rabbit limbal epithelial cells was also blocked under hypoxic conditions in corneal epithelial cell sheets engineered ex vivo. We concluded that hypoxia can prevent abnormal differentiation while enhancing the proliferation of corneal limbal epithelial cells. Hypoxia coupled with air exposure can be used in the tissue engineering of corneal limbal epithelium.     

PFN1 and integrin‐β1/mTOR axis involvement in cornea differentiation of fibroblast limbal stem cells

Ex vivo limbal stem cell transplantation is the main therapeutic approach to address a complete and functional re‐epithelialization in corneal blindness, the second most common eye disorder. Although important key points were defined, the molecular mechanisms involved in the epithelial phenotype determination are unclear. Our previous studies have demonstrated the pluripotency and immune‐modulatory of fibroblast limbal stem cells (f‐LSCs), isolated from the corneal limbus. We defined a proteomic profile especially enriched in wound healing and cytoskeleton‐remodelling proteins, including Profilin‐1 (PFN1). In this study we postulate that pfn‐1 knock down promotes epithelial lineage by inhibiting the integrin‐β1(CD29)/mTOR pathway and subsequent NANOG down‐expression. We showed that it is possible modulate pfn1 expression levels by treating f‐LSCs with Resveratrol (RSV), a natural compound: pfn1 decline is accompanied with up‐regulation of the specific differentiation epithelial genes pax6 (paired‐box 6), sox17 (sex determining region Y‐box 17) and ΔNp63‐α (p63 splice variant), consistent with drop‐down of the principle stem gene levels. These results contribute to understand the molecular biology of corneal epithelium development and suggest that pfn1 is a potential molecular target for the treatment of corneal blindness based on epithelial cell dysfunction.     

山羊角膜缘干细胞荧光蛋白(Venus)细胞株的建立及角膜上皮植片构建

角膜缘干细胞是角膜上皮更新与修复的来源,角膜上皮受损严重常会导致角膜盲。尽管近几年通过角膜缘干细胞移植术(LSCT)治愈角膜上皮受损的临床应用已被推广,但是对于角膜缘干细胞移植受损机体后的修复机理并不明确。为了实现角膜缘干细胞移植后的活体追踪,使用G418筛选标记有Venus荧光蛋白的角膜缘干细胞株(GLSC-V),并以其为种子细胞接种于去上皮羊膜上,体外培养21d构建成荧光角膜上皮植片。荧光倒置显微镜下观察GLSC-V的细胞质和细胞核均有绿色荧光表达,在体外培养荧光至少持续3个月。免疫荧光检测GLSC-V细胞P63、Integrinβ1均呈阳性表达,对GLSC-V细胞及未转染的GLSCs进行半定量RT-PCR检测显示,两组细胞皆未表达终末分化角膜上皮细胞基因k3、k12,GLSC-V中p63及pcna较未转染组细胞略上调,venus强表达。经HE染色观察构建的人工角膜组织由5～6层上皮细胞组成,组织中上表皮细胞个数少、体积大且呈扁平状;基底部细胞密集、体积小且成立方状。经免疫荧光检测仅组织基底部最基层细胞表达P63,上表皮细胞不表达。该人工角膜与正常角膜上皮组织结构特性相似,可用于移植,为研究角膜缘干细胞修复严重受损角膜上皮机理奠定基础。     

Ocular surface epithelial and stem cell development

Phenotypic features and developmental events involved in the genesis of the limbo-corneal and conjunctival epithelia are described. Together, these two epithelia define the ocular surface. They derive from a small cohort of optic vesicle-induced PAX6+ head ectodermal cells that remain on the surface following lens vesicle formation by the main PAX6+ cell cohort. Both epithelia are stratified, and display wet, non-keratinizing phenotypes. The most significant spatial feature of the limbo-corneal epithelium is the segregation of its supporting stem and early precursor cells to the limbus, the outer vascularized rim separating the cornea from the conjunctiva. These stem cells express ABCG2, a xenobiotic transporter present in stem cells from other organs. ABCG2 transport activity excludes the DNA dye Hoechst 33342, allowing the isolation of the ocular stem cells by flow cytometry, as a unique cohort known as a side 'side population'. Limbal stem cells do not form gap junctions and exist as metabolically isolated entities. Tracking of expression changes in Cx43, the main gap junction protein expressed in both the pre-epithelial ectoderm and in the mature central corneal epithelium, indicates that a limbal stem cell phenotype starts developing very soon after lens vesicle invagination, in advance of the appearance of any recognizable anatomical sub-epithelial limbal feature. Differences in Cx43 expression also reveal the very early nature of the divergence in limbo-corneal and conjunctival lineages. The putative involvement of several early genes, including gradients of PAX6 and differences in expression patterns for members of the Id or msh gene expression regulators are reviewed.     

Identification of human fibroblast cell lines as a feeder layer for human corneal epithelial regeneration

There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5-14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1 × 10(4) in a 35-mm dish (9.6 cm(2)) grew to confluence (about 1.87-2.41 × 10(6) cells) in 12-14 days, representing 187-241 fold expansion with over 7-8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction.     

Effects of different extracellular matrices and co-cultures on human limbal stem cell expansion in vitro

To elucidate the effect of extracellular matrices (ECMs) and related and nonrelated-limbal feeder cells as substitutes for the in vivo niche on the phenotype and genotype of the limbal stem cell (SC) expansion in vitro, human limbal SCs were used. The limbus explants were expanded on human amniotic membrane (AM), commercial ECMs including matrigel (MAT), collagen (COL), and control (no ECM) in presence and absence of feeder cells including human limbal fibroblasts (LFs), a limbus-specific cell and mouse embryonic fibroblasts (MEFs). Proliferation, cell death, immunocytochemistry, expression of specific genes, ultrastructural characteristics, and size and granularity of expanded human limbal SCs in different groups were evaluated. The growth, cell proliferation, and survival of limbal SCs were enhanced by AM and MAT matrices. Ultrastructure and expression of stemness markers revealed that there was no significance difference between AM and MAT. However, flow cytometric analysis showed that the size and granularity of cultured cells increased in the presence of MAT and COL as well as in no ECM group. Moreover, co-culturing of limbal explants with LFs and MEFs on AM and MAT groups, enhanced the expansion and survival of cultured cells in comparison with others. In conclusion, the cultivation of human limbal explants on AM co-culturing with human LFs promises to be a good model for preparing undifferentiated epithelial sheets suitable for transplantation.     

Subconjunctival Injection of Transdifferentiated Oral Mucosal Epithelial Cells for Limbal Stem Cell Deficiency in Rats

Rat limbal niche cells (LNCs) have been proven to induce transdifferentiation of oral mucosal epithelial cells (OMECs) into corneal epithelial-like cells termed transdifferentiated oral mucosal epithelial cells (T-OMECs). This investigation aimed to evaluate the effect of subconjunctival T-OMEC injections on alkali-induced limbal stem cell deficiency (LSCD) in rats. LNCs were cocultured with OMECs in the Transwell system to obtain T-OMECs, with NIH-3T3 cells serving as a control. Subconjunctival injection of single T-OMEC or OMEC suspension was performed immediately after corneal alkali injury. T-OMECs were prelabeled with the fluorescent dye CM-DiI in vitro and tracked in vivo. Corneal epithelial defect, opacity, and neovascularization were quantitatively analyzed. The degree of corneal epithelial defect (from day 1 onward), opacity (from day 5 onward), and neovascularization (from day 2 onward) was significantly less in the T-OMEC group than in the OMEC group. Cytokeratin 12 (CK12), pigment epithelium–derived factor, and soluble fms-like tyrosine kinase-1 were expressed at a higher rate following T-OMEC injection. Some CM-DiI-labeled cells were found to be coexpressed with CK12, Pax6, and ΔNp63α in the corneal epithelium after subconjunctival injection. Subconjunctival injection of T-OMECs prevents conjunctival invasion and maintains a normal corneal phenotype, which might be a novel strategy in the treatment of LSCD:     

Culture of limbal stem cells on human amniotic membrane

Limbal stem cells (LSC) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell (HLECs) transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. A major challenge is the ability to identify LSC in vitro and in situ, and one of the major controversies in the field relates to reliable LSC markers. This study was carried out to evaluate the culture of a limbal biopsy on human amniotic membrane (HAM): directly on the chorionic side and on intact epithelium, and the expression of the stem cell associated markers: ABCG2, p63. HAM has been extensively used for ocular surface reconstruction and has properties which facilitate the growth of epithelial cells controlling inflammation and scarring.     

A simple and efficient method for cultivation of limbal explant stem cells with clinically safe potential

Background

Several methods to cultivate limbal epithelial stem cells (LESCs) in vitro with the support of feeder layers and different growth medium formulations have been established for several years. The initial green medium consists of various ingredients that exhibit a non-optimal level of biosafety, therefore, different modifications have been made to suit it to safe clinical applications. However, the question of which formulation is the most appropriate remains to be answered.