Limbal Epithelial Stem Cells Markers Research Articles

FoxC1 activates limbal epithelial stem cells following corneal epithelial debridement

Limbal epithelial stem cells are not only critical for corneal epithelial homeostasis but also have the capacity to change from a relatively quiescent mitotic phenotype to a rapidly proliferating cell in response to population depletion following corneal epithelial wounding. Pax6+/− mice display many abnormalities including corneal vascularization and these aberrations are consistent with a limbal stem cell deficiency (LSCD) phenotype. FoxC1 has an inhibitory effect on corneal avascularity and a positive role in stem cell maintenance in many tissues. However, the role of FoxC1 in limbal epithelial stem cells remains unknown. To unravel FoxC1's role(s) in limbal epithelial stem cell homeostasis, we utilized an adeno-associated virus (AAV) vector to topically deliver human FOXC1 proteins into Pax6+/− mouse limbal epithelium. Under unperturbed conditions, overexpression of FOXC1 in the limbal epithelium had little significant change in differentiation (PAI-2, Krt12) and proliferation (BrdU, Ki67). Conversely, such overexpression resulted in a marked increase in the expression of putative limbal epithelial stem cell markers, N-cadherin and Lrig1. After corneal injuries in Pax6+/− mice, FOXC1 overexpression enhanced the behavior of limbal epithelial stem cells from quiescence to a highly proliferative status. Overall, the treatment of AAV8-FOXC1 may be beneficial to the function of limbal epithelial stem cells in the context of a deficiency of Pax6 function.

Pax6-Induced Proliferation and Differentiation of Bone Marrow Mesenchymal Stem Cells Into Limbal Epithelial Stem Cells.

Corneal integrity, transparency, and visual acuity are maintained by corneal epithelial cells (CECs), which are continuously renewed by limbal epithelial stem cells (LESCs). The limbal stem cell deficiency is associated with ocular diseases. This study aimed to develop a novel method to differentiate bone marrow mesenchymal stem cells (BM-MSCs) into LESC-like cells using a culture medium and paired box 6 (Pax6) transfection. The LESC-like cells were confirmed using the LESC markers CK14 and p63 and CEC marker CK12. Pax6 induces BM-MSCs to differentiate into LESC-like cells in vitro. Mouse models of chemical corneal burn were obtained and treated with the LESC-like cells. The transplantation experiment indicated that Pax6-reprogrammed BM-MSCs attached to and replenished the damaged cornea through the formation of stratified corneal epithelium. The proliferation and colony formation abilities of Pax6-overexpressing BM-MSCs were significantly enhanced. These findings provide evidence that BM-MSCs might serve as an excellent candidate for generating bioengineered corneal epithelium and provide a new strategy for the treatment of clinical corneal damage.

Corneal Repair with Adhesive Cell Sheets of Fetal Cartilage-Derived Stem Cells.

Corneal scarring or disease may lead to severe corneal opacification and consequently, severe loss of vision due to the complete loss of corneal epithelial cells. We studied the use of epithelial cell sheets differentiated from fetal cartilage-derived stem cells (FCSC) to resurface damaged cornea. The FCSC were isolated from the femoral head of immature cartilage tissue. The ability of the FCSCs to differentiate into corneal epithelial cells was evaluated using differentiation media at 2days and 7days post-seeding. A sheet fabricated of FCSCs was also used for the differentiation assay. The results of the in vitro studies were evaluated by immunocytochemistry and Western blots for corneal epithelial cell markers (CK3/12 and Pax6) and limbal epithelial stem cell markers (ABCG2 and p63). To test the material in vivo, an FCSC-sheet was applied as a treatment in a chemically burned rabbit model. The healing ability was observed histologically one week after treatment. The in vitro experiments showed morphological changes in the FCSCs at two and seven days of culture. The differentiated cells from the FCSCs or the FCSC-sheet expressed corneal epithelial cells markers. FCSC were create cell sheet that successfully differentiated into corneal epithelial cells and had sufficient adhesion so that it could be fused to host tissue after suture to the ocular surface with silk suture. The implanted cell sheet maintained its transparency and the cells were alive a week after implantation. These results suggest that carrier-free sheets fabricated of FCSCs have the potential to repair damaged corneal surfaces.

YAP, ΔNp63, and β-Catenin Signaling Pathways Are Involved in the Modulation of Corneal Epithelial Stem Cell Phenotype Induced by Substrate Stiffness.

Recent studies have established that the phenotype of epithelial stem cells residing in the corneal periphery (the limbus) depends on this niche’s distinct biomechanical properties. However, the signaling pathways underlying this dependency are still poorly understood. To address this issue, we investigated the effect of substrate stiffness on the migration, proliferation, and molecular phenotype of human limbal epithelial stem cells (LESCs). Specifically, we demonstrated that cells grown on collagen-based substrates with limbus-like compliance showed higher proliferation and stratification and lower migration capabilities, as well as higher levels of pro-proliferative markers Ki67 and β-Catenin, and LESC markers ΔNp63, ABCG2, and CK15. In contrast, cells on stiffer substrates lost these stem/progenitor cell markers, but instead expressed the key mechanotransduction factor YAP, as well as elevated levels of BMP4, a promotor of cell differentiation known to be negatively regulated by Wnt/β-Catenin signaling. This data allowed us to propose a new model that integrates the various molecular pathways involved in LESC response to substrate stiffness. This model will potentially be a useful guide to future research on the mechanisms underlying LESC loss following fibrosis-causing injuries.

In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds.

Purpose To investigate the efficacy of recombinant human collagen type I (RHC I) and collagen-like peptide (CLP) hydrogels as alternative carrier substrates for the cultivation of limbal epithelial stem cells (LESC) under xeno-free culture conditions. Methods Human LESC were cultivated on seven different collagen-derived hydrogels: (1) unmodified RHC I, (2) fibronectin-patterned RHC I, (3) carbodiimide-crosslinked CLP (CLP-12 EDC), (4) DMTMM- (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium-) crosslinked CLP (CLP-12), (5) fibronectin-patterned CLP-12, (6) “3D limbal niche-mimicking” CLP-12, and (7) DMTMM-crosslinked CLP made from higher CLP concentration solution. Cell proliferation, cell morphology, and expression of LESC markers were analyzed. All data were compared to cultures on human amniotic membrane (HAM). Results Human LESC were successfully cultivated on six out of seven hydrogel formulations, with primary cell cultures on CLP-12 EDC being deemed unsuccessful since the area of outgrowth did not meet quality standards (i.e., inconsistence in outgrowth and confluence) after 14 days of culture. Upon confluence, primary LESC showed high expression of the stem cell marker ΔNp63, proliferation marker cytokeratin (KRT) 14, adhesion markers integrin-β4 and E-cadherin, and LESC-specific extracellular matrix proteins laminin-α1, and collagen type IV. Cells showed low expression of differentiation markers KRT3 and desmoglein 3 (DSG3). Significantly higher gene expression of KRT3 was observed for cells cultured on CLP hydrogels compared to RHC I and HAM. Surface patterning of hydrogels influenced the pattern of proliferation but had no significant effect on the phenotype or genotype of cultures. Overall, the performance of RHC I and DMTMM-crosslinked CLP hydrogels was equivalent to that of HAM. Conclusion RHC I and DMTMM-crosslinked CLP hydrogels, irrespective of surface modification, support successful cultivation of primary human LESC using a xeno-free cultivation protocol. The regenerated epithelium maintained similar characteristics to HAM-based cultures.

Exosomes from normal and diabetic human corneolimbal keratocytes differentially regulate migration, proliferation and marker expression of limbal epithelial cells

Limbal epithelial stem cells (LESC) maintenance requires communication between stem cells and neighboring stromal keratocytes. Extracellular vesicles (EVs) are important for intercellular communication in various stem cell niches. We explored the regulatory roles of limbal stromal cell (LSC)-derived exosomes (Exos), an EV sub-population, in limbal epithelial cells (LEC) in normal and diabetic limbal niche and determined differences in Exo cargos from normal and diabetic LSC. Wound healing and proliferation rates in primary normal LEC were significantly enhanced upon treatment by normal Exos (N-Exos), but not by diabetic Exos (DM-Exos). Western analysis showed increased Akt phosphorylation in wounded LECs and organ-cultured corneas treated with N-Exos, compared to untreated wounded cells and DM-Exos treated fellow corneas, respectively. N-Exos treated organ-cultured corneas showed upregulation of putative LESC markers, keratin 15 (K15) and Frizzled-7, compared to the DM-Exos treated fellow corneas. By next generation sequencing, we identified differentially expressed small RNAs including microRNAs in DM-Exos vs. N-Exos. Overall, N-Exos have greater effect on LEC proliferation and wound healing than DM-Exos, likely by activating Akt signaling. The small RNA differences in Exos from diabetic vs. normal LSC could contribute to the disease state. Our study suggests that exosomes may serve as novel therapeutic tools for diabetic cornea.

Comparison of culture media indicates a role for autologous serum in enhancing phenotypic preservation of rabbit limbal stem cells in explant culture.

In this study, we aimed to compare the effects of six different cell culture media and autologous serum (AS) on the phenotypic characteristics of rabbit limbal epithelial stem cells (LESC) cultivated on porous polyethylene terephthalate (PET) membranes. Limbal explants from rabbit corneas were grown on PET membrane inserts in five different media: DMEM-F12 with fetal bovine serum (FBS) (DMEM-F12-FBS), with pluripotin (DMEM-F12-pluripotin) and with autologous serum (DMEM-F12-AS), Epilife, Keratinocyte Serum Free Medium (KSFM) and Defined-Keratinocyte Serum Free Medium. The effects of different media were evaluated by total cell yield from explants, measuring the expression of proteins by immunofluorescence and gene expression by Real Time PCR. In all five media tested, most of the limbal epithelial cells (LEC) which proliferated from explants were positive for cytokeratin (CK) 14 (85-90%), indicating that all five media support the growth of LESC from explants. The expression of differentiation markers; CK 3 and 12 was highest in DMEM-F12-FBS (56%), was lower in Epilife and KSFM (26 and 19%, respectively), with the lowest values (13%) obtained in DMEM-F12-AS. Gene expression of limbal cultures on PET membrane inserts was compared to fresh limbal tissue. In DMEM-F12-FBS, DMEM-F12-pluripotin, and DMEM-F12-AS, expression of potential LESC markers CXCR4 and polycomb complex protein BMI-1 were similar to limbal tissue. DMEM-F12 with 10% AS maintained a higher percentage of potential stem cell marker genes and lower expression of genes involved in differentiation compared to Epilife or KSFM. Our study shows that rabbit LEC can be cultivated on PET inserts using DMEM-F12 with autologous serum without a requirement for amniotic membrane or feeder cells.

Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method

BackgroundHuman pluripotent stem cells (hPSCs) provide a promising cell source for ocular cell replacement therapy, but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and feeder cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target cells: retinal pigment epithelial (RPE) cells and corneal limbal epithelial stem cells (LESCs).MethodsMultiple genetically distinct hPSC lines were adapted to a defined, xeno-, and feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial cells were established utilizing xeno-free media and a combination of extracellular matrix proteins. Both differentiation methods shared the same basal elements, using only minor inductive modifications during early differentiation towards desired cell lineages. The resulting RPE cells and LESCs were characterized after several independent differentiation experiments and recovery after xeno-free cryopreservation.ResultsThe defined, xeno-, and feeder-free culture system provided a robust means to generate high-quality hPSCs with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation had a substantial effect on lineage specification, cell survival, and even mature RPE properties. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE cells, and hPSC-LESCs, and demonstrated successful recovery after thawing.ConclusionsWe propose methods for efficient and scalable, directed differentiation of high-quality RPE cells and LESCs. The two clinically relevant cell types are generated with simple inductive modification of the same basal method, followed by adherent culture, passaging, and cryobanking.

Limbal and Conjunctival Epithelial Cell Cultivation on Contact Lenses-Different Affixing Techniques and the Effect of Feeder Cells.

Corneal blindness due to limbal stem-cell deficiency can be treated by transplantation of cultivated limbal epithelial stem cells (LESCs). We examined LESC cultivation on a contact lens (CL) carrier. Our goal was to optimize explant affixation and assess the possible benefit of 3T3 feeder cells. Human cadaver limbal and conjunctival explants were allowed to attach to CLs under the airflow of the laminar box (dried group) or affixed on CLs using suturing (sutured group) or tissue adhesives (glued group), then cultivated with or without 3T3 feeder cells. Outgrowth efficiency was statistically analyzed. CEBPδ, p63, CK3/12, and CK13 were detected by immunofluorescence in expanded cells. Suturing and gluing provided excellent sample attachment, whereas drying was less effective. Cell expansion was better in sutured than in dried or glued samples. Presence of 3T3 feeder resulted in significantly better cell growth (P=0.048), most importantly in dried samples (P=0.008). Stepwise regression analysis indicated that cell expansion was dependent on the affixing method (P<0.001) and the presence of feeder layer (P=0.003). Expanded cells maintained their CK expression profiles and expressed putative stem-cell markers p63 and CEBPδ. The 3T3 feeder did not influence the expression of putative LESC markers or growth rate. Suturing is an effective way to fasten explants to CLs. 3T3 fibroblasts are not necessary in this system, although they may enhance cell outgrowth when samples are exposed to stress. However, once cells begin to expand, neither expression of putative stem-cell markers nor growth rate is influenced by feeder cells.

Successful Consecutive Expansion of Limbal Explants Using a Biosafe Culture Medium under Feeder Layer-Free Conditions

ABSTRACTPurpose: Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers.Materials and methods: We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence.Results: LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished.Conclusion: IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure.

Corneal Epithelial Stem Cells Repopulate the Donor Area within 1 Year from Limbus Removal for Limbal Autograft

To determine whether limbal epithelial stem cells (LESCs) repopulate the site harvested for limbal autograft transplantation (LAT), the expression of LESCs markers was evaluated in bioptic specimens obtained from the donor area 12 months or more after surgery. Interventional case series. Patients who underwent LAT for unilateral acquired limbal stem cell deficiency after chemical burn. Corneal limbal explants were obtained from 2 sites, the harvested area and the untouched control area, in the donor eyes of 6 patients who previously underwent LAT for unilateral acquired limbal stem cell deficiency after chemical burn. Limbal epithelial stem cells were isolated, and cellular, immunohistochemistry, and histologic parameters were assessed to compare differences between LESCs isolated from harvested or control sites. Presence of LESCs 1 year or more after LAT. Specific markers (p63, Ki67, K12), percentage of LESCs, cell doubling, and number of passages in culture did not differ significantly between harvested and control sites. However, the distinctive structure of the palisades of Vogt was found only in 2 of 6 harvested sites. Limbal epithelial stem cells repopulate the donor site as early as 1 year after limbus removal for LAT. Autologous transplantation of conjunctiva and limbus are safe procedures and can be performed in cases that cannot be treated by simple grafting of LESCs cultured exvivo.

Recreating the Human Limbal Epithelial Stem Cell Niche with Bioengineered Limbal Crypts

ABSTRACTPurpose: Creation of an in vitro model incorporating specific features that characterize a particular stem niche would allow researchers to study stem cell behavior in a more physiological environment.Materials and methods: We have developed a tissue engineering process (RAFT) that rapidly and reliably creates bioengineered limbal crypts (BLCs) in the surface of collagen-based tissue equivalents (TEs). These BLCs mimic the three-dimensional topography of the limbal crypts (LCs), located in the limbal region of the human cornea, which are home to a population of limbal epithelial stem cells (LESCs).Results: Human limbal epithelial (hLE) cells occupying our BLCs expressed putative LESC markers such as ΔNp63α and Bmi1 and produced basement membrane proteins such as laminin β1 and laminin γ3; expression patterns are very similar to those seen in native LCs. Human limbal stromal cells elongate and align along the edge of native LCs and in our RAFT TEs, human limbal fibroblasts (hLFs) also appeared to exhibit this alignment and elongation behavior in response to the BLC topography.Conclusions: We have demonstrated that we can maintain an immature population of hLE cells and aligned stromal cells in our BLCs to mimic some elements of the complexity of the human LESC niche.

Comparison of functional limbal epithelial stem cell isolation methods

The transplantation of limbal epithelial stem cells (LESCs) cultured in vitro is a great advance in the treatment of patients suffering from LESC deficiency. However, the optimal technique for LESC isolation from a healthy limbal niche has not yet been established. Our aim was to determine which isolation method renders the highest recovery of functional LESCs from the human limbus. To achieve this purpose, we compared limbal primary cultures (LPCs) obtained from explants and cell suspensions on plastic culture plates. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC, corneal epithelial cell, fibroblast, endothelial cell, melanocyte, and dendritic cell markers were analyzed by real time by reverse transcription polymerase chain reaction and/or immunofluorescence. In addition, colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were studied. We observed that LPC cells obtained from both methods had cuboidal morphology, desmosomes, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPCs established through cell suspensions, except the expression of p63α mRNA, and there were no significant differences in the expression of corneal epithelial markers (K3, K12). Endothelial cell (PECAM), melanocyte (MART-1), and dendritic cell (CD11c) proteins were not detected, while fibroblast-protein (S100A4) was detected in all LPCs. The CFE was significantly higher in LPCs from cell suspensions. Cells from confluent LPCs produced by explants generated only paraclones (100%), while the percentage of paraclones from LPCs established through cell suspensions was 90% and the remaining 10% were meroclones. In conclusion, LPCs established from cell suspensions have a cell population richer in functional LESCs than LPCs obtained from explants. These results suggest that in a clinical situation in which it is possible to choose between either of the isolation techniques from the donor limbal tissue, then the cell suspension is probably the best option as long as the cells are expanded following our culture conditions.

Human pluripotent stem cell-derived limbal epithelial stem cells on bioengineered matrices for corneal reconstruction

Corneal epithelium is renewed by limbal epithelial stem cells (LESCs), a type of tissue-specific stem cells located in the limbal palisades of Vogt at the corneo-scleral junction. Acute trauma or inflammatory disorders of the ocular surface can destroy these stem cells, leading to limbal stem cell deficiency (LSCD) – a painful and vision-threatening condition. Treating these disorders is often challenging and complex, especially in bilateral cases with extensive damage. Human pluripotent stem cells (hPSCs) provide new opportunities for corneal reconstruction using cell-based therapy. Here, we investigated the use of hPSC-derived LESC-like cells on bioengineered collagen matrices in serum-free conditions, aiming for clinical applications to reconstruct the corneal epithelium and partially replace the damaged stroma. Differentiation of hPSCs towards LESC-like cells was directed using small-molecule induction followed by maturation in corneal epithelium culture medium. After four to five weeks of culture, differentiated cells were seeded onto bioengineered matrices fabricated as transparent membranes of uniform thickness, using medical-grade porcine collagen type I and a hybrid cross-linking technology. The bioengineered matrices were fully transparent, with high water content and swelling capacity, and parallel lamellar microstructure. Cell proliferation of hPSC-LESCs was significantly higher on bioengineered matrices than on collagen-coated control wells after two weeks of culture, and LESC markers p63 and cytokeratin 15, along with proliferation marker Ki67 were expressed even after 30 days in culture. Overall, hPSC-LESCs retained their capacity to self-renew and proliferate, but were also able to terminally differentiate upon stimulation, as suggested by protein expression of cytokeratins 3 and 12. We propose the use of bioengineered collagen matrices as carriers for the clinically-relevant hPSC-derived LESC-like cells, as a novel tissue engineering approach for corneal reconstruction.

Differentiation of human limbal-derived induced pluripotent stem cells into limbal-like epithelium.

Limbal epithelial stem cell (LESC) deficiency (LSCD) leads to corneal abnormalities resulting in compromised vision and blindness. LSCD can be potentially treated by transplantation of appropriate cells, which should be easily expandable and bankable. Induced pluripotent stem cells (iPSCs) are a promising source of transplantable LESCs. The purpose of this study was to generate human iPSCs and direct them to limbal differentiation by maintaining them on natural substrata mimicking the native LESC niche, including feederless denuded human amniotic membrane (HAM) and de-epithelialized corneas. These iPSCs were generated with nonintegrating vectors from human primary limbal epithelial cells. This choice of parent cells was supposed to enhance limbal cell differentiation from iPSCs by partial retention of parental epigenetic signatures in iPSCs. When the gene methylation patterns were compared in iPSCs to parental LESCs using Illumina global methylation arrays, limbal-derived iPSCs had fewer unique methylation changes than fibroblast-derived iPSCs, suggesting retention of epigenetic memory during reprogramming. Limbal iPSCs cultured for 2 weeks on HAM developed markedly higher expression of putative LESC markers ABCG2, ΔNp63α, keratins 14, 15, and 17, N-cadherin, and TrkA than did fibroblast iPSCs. On HAM culture, the methylation profiles of select limbal iPSC genes (including NTRK1, coding for TrkA protein) became closer to the parental cells, but fibroblast iPSCs remained closer to parental fibroblasts. On denuded air-lifted corneas, limbal iPSCs even upregulated differentiated corneal keratins 3 and 12. These data emphasize the importance of the natural niche and limbal tissue of origin in generating iPSCs as a LESC source with translational potential for LSCD treatment.

Consecutive Expansion of Limbal Epithelial Stem Cells from a Single Limbal Biopsy

Purpose: Corneal epithelium is maintained by limbal epithelial stem cells (LESCs), the loss of which can be catastrophic for corneal transparency. Effective therapies include the transplantation of cultivated LESCs, requiring optimization of in vitro cultivation protocols. Unfortunately, optimization studies are hampered by the limited number of ocular tissue donors. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same 1-2 mm2 limbal explant (LE).Methods: LEs were plated and maintained until outgrowth surrounded each, being removed at this point. LPCs were allowed to reach confluence (LPC0). The same removed LE was plated again, following the same procedure, obtaining LPC1. This procedure was repeated as often as possible up to six times. LPCs from each passage were analysed by real time reverse transcription-polymerase chain reaction and immunofluorescence-microscopy.Results: LPCs from LPC0 to LPC2 presented a heterogeneous cell population, with cells positive for LESC markers K14, K15, ABCG2 and p63, differentiated corneal epithelial cell-specific markers K3 and K12, and for the fibroblast marker S100A4. These cells had an epithelial-like morphology. In LPC3-LPC4, elongated cell morphology appeared, and the presence of LESC markers decreased, while the presence of differentiated corneal epithelial-cell and fibroblast markers increased.Conclusion: One LE can be successfully cultivated up to three consecutive times while maintaining the LESC phenotype in the LPC cells. This protocol provides several homologous LPCs for basic research. Additionally, by using a cell-carrier, the resulting LPCs could serve reservoirs for potential autologous expanded LESC transplantations and/or for making correlations between laboratory and clinical outcomes.

Expression of human limbal epithelial stem cells markers and mRNA in long term repeated limbal cultures

Expression of human limbal epithelial stem cells markers and mRNA in long term repeated limbal cultures

Effect of Sub-Atmospheric Oxygen on the Culture of Rabbit Limbal Epithelial Cells

Purpose: To investigate the effect of sub-atmospheric oxygen tensions on the maintenance and expansion of limbal epithelial cells in vitro.Materials and Methods: Limbal epithelial cells were isolated from rabbit corneas and cultured in 21%, 14%, 8%, and 2% oxygen in a co-culture system with Mitomycin C growth arrested 3T3 fibroblasts. The colony forming efficiency, proliferative ability, and cell cycle distribution of cells cultured in these different oxygen tensions were determined, and semi-quantitative RT-PCR was used to detect expression of putative limbal epithelial stem cell markers, such as ABCG2 and p63α.Results: Of the four different oxygen tensions studied, 14% and 2% supported the highest and lowest colony forming efficiency values, respectively. A greater proportion of cells were found in S and G2/M phases of the cell cycle in primary 14% and 8% oxygen cultures compared to atmospheric controls. Differentiation was promoted at oxygen tensions of 8% and below. Cells with a differentiated phenotype and limited proliferative capacity were observed in 2% oxygen. Semi-quantitative RT-PCR analysis showed that the putative limbal epithelial stem cell markers ABCG2 and p63α were expressed in 21%, 14%, and 8% oxygen, with a trend of lower expression in 8% oxygen being observed.Conclusions: Limbal epithelial cells are sensitive to in vitro changes in oxygen tension. A sub-atmospheric oxygen tension of 14% promoted the maintenance and expansion of cells with limbal epithelial stem cell characteristics in a feeder co-culture system and is, therefore, recommended for the culture of rabbit limbal epithelial cells. This may also have relevance for the culture of human limbal epithelial stem cells for therapeutic application.

Molecular and cellular characterization of expanded and cryopreserved human limbal epithelial stem cells reveal unique immunological properties

Transplantation of ex vivo expanded autologous limbal stem cells into the diseased eye of patients with limbal stem cell deficiency (LSCD) has been in practice worldwide. However, isolation of limbal tissue from the normal eye of the patient with unilateral LSCD still remains a major concern for the donor. More importantly, autologous cell transplantation is not a viable option for patients with bilateral LSCD. The objective of the current study was to determine the expansion potential of human limbal epithelial stem cells (hLESCs) for their possible use in allo-transplantation. A total of six limbal biopsy samples were cultured and expanded in vitro up to passage level 1 (P-1), at which point the hLESCs were cryopreserved. Semi-quantitative RT-PCR and immunophenotypic analysis revealed that hLESCs obtained before and after cryopreservation retained the expression of major limbal epithelial stem cell markers such as p63, SSEA-4, ABCG2, cytokeratin 19 (CK19), integrin β1 and vimentin. One notable difference was that while P-0 hLESCs expressed HLA-DR mRNA, no HLA-DR gene expression was observed with the expanded and cryopreserved samples. Human LESCs did not express costimulatory proteins CD80 or B7-DC but expressed significant levels of CD86, B7-H1 and HLA-ABC molecules on the cell surface. Treatment of hLESCs with IFN-γ induced the expression of HLA-DR, indoleamine 2,3-dioxygenase (IDO) and HLA-G on these cells. Cultured hLESCs were unable to stimulate allogeneic T cell proliferation in vitro even in the presence of pro-inflammatory cytokine, IFN-γ. These results indicate that cryopreserved hLESCs are non-immunogenic in nature and express negative immunoregulatory molecules which may be critical for their survival in an allogeneic environment.