A 3D Polymer Scaffold Platform for Enhanced in vitro Culture of Human & Rabbit Buccal Epithelial Cells for Cell Therapies.

BACKGROUND: Buccal mucosal epithelial cells show promising application for various regenerative medicine approaches. In this study, we examined the feasibility of culturing rabbit and human buccal mucosal epithelial cells in a novel thermoreversible gelation polymer (TGP) scaffold, without feeder layers or other foreign proteins. METHODS & RESULTS: The results of this 28-day in vitro culture, u sing the conventional technique (2D) and TGP (3D) showed that the epithelial cell morphology could be maintained only in the TGP group while cells in the 2D group de-differentiated to fibroblast morphology in both human and rabbit samples. CK3 expression, a marker for epithelial differentiation was higher in 3D-TGP cultured cells than 2D. CONCLUSION: TGP based in vitro cell culture is a prospective methodology to culture buccal mucosal epithelial cells efficiently without using foreign biological components for tissue engineering applications.

Coexistence of Meesmann Corneal Dystrophy and a Pseudo-Unilateral Lattice Corneal Dystrophy in a Patient With a Novel Pathogenic Variant in the Keratin K3 Gene: A Case Report.

PURPOSE: This study aims to clinically and genetically report a case of coexisting Meesmann corneal dystrophy (MECD) and pseudo-unilateral lattice corneal dystrophy (LCD). METHODS: Clinical characterization was supported by a complete ophthalmological evaluation, including visual acuity measurement and slit-lamp examination. Molecular diagnosis was performed by whole-exome sequencing analyzing the gelsolin, keratin K3 (KRT3), keratin K12, and transforming growth factor-beta-induced genes. RESULTS: A 57-year-old woman presented with recurrent corneal erosions over 17 years and visual impairment in both eyes. Ophthalmological evaluation revealed multiple central tiny cysts in the epithelium of both eyes and lattice linear lesions only in the right cornea. In both eyes, a corneal posterior crocodile shagreen degeneration could also be observed. These findings were compatible with a MECD and a unilateral LCD. Molecular analysis identified the novel heterozygous nucleotide substitution c.1492G>A (amino acid change p.Glu498Lys) in the KRT3 gene, in cosegregation with the MECD familial phenotype. However, no genetic evidence supported the unique LCD phenotype observed in the patient. CONCLUSIONS: To the best of our knowledge, this is the first report of a pseudo-unilateral LCD in a patient with coexistent MECD. Moreover, the genetic analysis showed a novel mutation in the previously MECD-associated gene KRT3.

Xenofree generation of limbal stem cells for ocular surface advanced cell therapy.

BACKGROUND: Limbal stem cells (LSC) sustain the corneal integrity and homeostasis. LSC deficiency (LSCD) leads to loss of corneal transparency and blindness. A clinical approach to treat unilateral LSCD comprises autologous cultured limbal epithelial stem cell transplantation (CLET). CLET uses xenobiotic culture systems with potential zoonotic transmission risks, and regulatory guidelines make necessary to find xenofree alternatives. METHODS: We compared two xenofree clinical grade media and two feeder layers. We used CnT07, a defined commercial medium for keratinocytes, and a modified xenofree supplemented hormonal epithelial medium with human serum (XSHEM). Optimal formulation was used to compare two feeder layers: the gold standard 3T3 murine fibroblasts and human processed lipoaspirate cells (PLA). We tested the expressions of ΔNp63α and cytokeratin 3 and 12 by qPCR and immunofluorescence. Morphology, viability, clonogenicity, proliferation, and cell growth assays were carried out. We also evaluated interleukin 6 (IL-6) and stromal-derived factor 1 (SDF-1) by qPCR and ELISA. RESULTS: XSHEM maintained better LSC culture viability and morphology than CnT07. Irradiated PLA feeder cells improved the undifferentiated state of LSC and enhanced their growth and clonogenicity stimulating IL-6 secretion and SDF-1 expression, as well as increased proliferation and cell growth when compared with irradiated 3T3 feeder cells. CONCLUSIONS: The combination of XSHEM and PLA feeder cells efficiently sustained LSC xenofree cultures for clinical application. Moreover, PLA feeder layers were able to improve the LSC potential characteristics. Our results would have direct clinical application in CLET for advanced therapy.

Human aniridia limbal epithelial cells lack expression of keratins K3 and K12.

Aniridia is a rare disease of the eye that affects the iris, lens and the cornea. In about 90% of the cases, patients showed a loss of PAX6 function. Patients with aniridia often develop aniridia-related keratopathy (ARK), due to limbal stem cell insufficiency. The aim of this study was to determine the differentiation status of limbal epithelial cells (LECs) in patients with ARK. Epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in KSFM medium supplemented with EGF and BPE. Normal cells were obtained from limbus region of cadaveric control patients. Cells were analyzed with RT-PCR, qPCR and Western blot to evaluate expression of the developmental transcription factor, PAX6, potential stem cell markers, ΔNp63α and ABCG2, and corneal differentiation markers, keratin 12 (K12) and K3. Conjunctival differentiation markers, keratin 13 (K13) and K19 were also investigated. Cells were immunostained to evaluate K3, PAX6, and p63α protein expression. Protein coding sequence of PAX6 from patient LEC-cDNA was cloned and sequenced. RT-PCR showed that K3 and K12 transcripts were absent from patient cells, but present in healthy control preparations. Transcription levels of PAX6, ABCG2, and p63α of aniridia patients show no differences compared to normal control cells. Western blot showed reduced PAX6, protein levels in aniridia-LECs compared to control-LECs. Immunostaining also showed reduced PAX6 and K3 expression in aniridia-LECs compared to control-LECs. One aniridia patient showed a loss of stop codon in half of the cloned transcripts. In the second aniridia patient mRNA degradation through nonsense mediated decay seems to be very likely since we could not identify the mutation c.174C > T (Refseq. NM_000280), or misspliced transcripts in cDNA. We identified decreased PAX6 protein levels in aniridia patients in addition to decreased K12 mRNA levels compared to control cells. This result indicates an altered differentiation of limbal epithelial cells of aniridia patients. Further studies are necessary to evaluate the mechanism of differentiation of limbal epithelial cells in aniridia.

Corneal regeneration by induced human buccal mucosa cultivated on an amniotic membrane following alkaline injury.

Various clinical disorders and injuries, such as chemical, thermal, or mechanical injuries, may lead to corneal loss that results in blindness. PURPOSE: The aims of this study were to differentiate human buccal mucosa (BMuc) into corneal epithelial-like cells, to fabricate engineered corneal tissue using buccal mucosal epithelial cells, and to reconstruct a damaged corneal epithelium in a nude rat model. Methods: BMuc were subjected to 10 d of induction factors to investigate the potential of cells to differentiate into corneal lineages. Results: Corneal stem cell markers ß1-integrin, C/EBPδ, ABCG2, p63, and CK3 were upregulated in the gene expression analysis in induced BMuc, whereas CK3 and p63 showed significant protein expression in induced BMuc compared to the uninduced cells. BMuc were then left to reach 80% confluency after differential trypsinization. The cells were harvested and cultivated on a commercially available untreated air-dried amniotic membrane (AM) in a Transwell system in induction medium. The corneal constructs were fabricated and then implanted into damaged rat corneas for up to 8 weeks. A significant improvement was detected in the treatment group at 8 weeks post-implantation, as revealed by slit lamp biomicroscopy analysis. The structure and thickness of the corneal layer were also analyzed using histological staining and time-domain optical coherence tomography scans and were found to resemble a native corneal layer. The protein expression for CK3 and p63 were continuously detected throughout the corneal epithelial layer in the corneal construct. Conclusions: In conclusion, human BMuc can be induced to express a corneal epithelial-like phenotype. The addition of BMuc improves corneal clarity, prevents vascularization, increases corneal thickness and stromal alignment, and appears to have no adverse effect on the host after implantation.

Sphere-forming cells from peripheral cornea demonstrate the ability to repopulate the ocular surface.

BACKGROUND: The limbus forms the outer rim of the cornea at the corneoscleral junction and harbours a population of stem cells for corneal maintenance. Injuries to the limbus, through disease or accidents such as chemical injuries or burns, may lead to significant visual impairment due to depletion of the native stem cells of the tissue. METHODS: Sphere-forming cells were isolated from peripheral cornea for potential use as transplantable elements for limbal stem cell repopulation and limbal reconstruction. Immunocytochemistry, live cell imaging and quantitative PCR were used to characterize spheres and elucidate activity post implantation into human cadaveric corneal tissue. RESULTS: Spheres stained positively for stem cell markers ∆NP63α, ABCG2 and ABCB5 as well as the basal limbal marker and putative niche marker, notch 1. In addition, spheres also stained positively for markers of corneal cells, vimentin, keratin 3, keratocan and laminin, indicating a heterogeneous mix of stromal and epithelial-origin cells. Upon implantation into decellularized corneoscleral tissue, 3D, polarized and radially orientated cell migration with cell proliferation was observed. Cells migrated out from the spheres and repopulated the entire corneal surface over 14 days. Post-implantation analysis revealed qualitative evidence of stem, stromal and epithelial cell markers while quantitative PCR showed a quantitative reduction in keratocan and laminin expression indicative of an enhanced progenitor cell response. Proliferation, quantified by PCNA expression, significantly increased at 4 days subsequently followed by a decrease at day 7 post implantation. CONCLUSION: These observations suggest great promise for the potential of peripheral corneal spheres as transplantable units for corneal repair, targeting ocular surface regeneration and stem cell repopulation.

PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes.

PAX6 is the key transcription factor involved in eye development in humans, but the differential functions of the two PAX6 isoforms, isoform-a and isoform-b, are largely unknown. To reveal their function in the corneal epithelium, PAX6 isoforms, along with reprogramming factors, were transduced into human non-ocular epithelial cells. Herein, we show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly keratin 3 (KRT3) and keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4, and OCT4 induced KRT12 expression. These new findings will contribute to furthering the understanding of the molecular basis of the corneal epithelium specific phenotype.

Keratin 12 missense mutation induces the unfolded protein response and apoptosis in Meesmann epithelial corneal dystrophy.

Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations.

Cellular Response of Limbal Stem Cells on Polycaprolactone Nanofibrous Scaffolds for Ocular Epithelial Regeneration.

PURPOSE: The aim of this study was to develop nanofibrous polycaprolactone (PCL) substrate for limbal stem cell (LSC) expansion that can serve as a potential alternative substrate to replace human amniotic membrane (AM). MATERIALS AND METHODS: The human limbus stem cell was used to evaluate the biocompatibility of substrates (nanofibrous scaffold and, human AM) based on their phenotypic profile, viability, proliferation and attachment ability. RESULTS: Biocompatibility results indicated that the all substrates were highly biocompatible, as LSCs could favorably attach and proliferate on the nanofibrous surface. Microscopic figures showed that the human LSCs were firmly anchored to the substrates and were able to retain a normal corneal stem cell phenotype. Microscopic analyses illustrated that cells infiltrated the nanofibers and successfully formed a three-dimensional corneal epithelium, which was viable for two weeks. Immunocytochemistry (ICC) and real time-PCR results revealed no change in the expression profile of LECs grown on nanofibrous substrate when compared to those grown on human AM. CONCLUSION: In addition, electrospun nanofibrous PCL substrate provides not only a milieu supporting LSCs expansion, but also serve as a useful alternative carrier for ocular surface tissue engineering and could be used as an alternative substrate to AM.

Differentiation of Stem Cells From Human Exfoliated Deciduous Teeth Toward a Phenotype of Corneal Epithelium In Vitro.

PURPOSE: The aim of this study was to characterize stem cells from human exfoliated deciduous teeth (SHED) and to investigate the potential of SHED to differentiate toward corneal epithelium-like cells in vitro. METHODS: Mesenchymal and embryonic stem cell markers were analyzed by flow cytometry. The SHED was cocultured in either a transwell noncontact system or in a mixed culture system with immortalized human corneal epithelial (HCE-T) cells to induce the epithelial transdifferentiation. Expression of the mature corneal epithelium-specific marker cytokeratin 3 (CK3) and corneal epithelial progenitor marker cytokeratin 19 (CK19) were detected by immunofluorescence and the reverse transcription-polymerase chain reaction, respectively. RESULTS: SHED strongly expressed a set of mesenchymal stromal cell markers and pluripotency markers including NANOG and OCT-4. Seven days after the transwells were cocultured with HCE-T cells, SHED successfully upregulated epithelial lineage markers CK3 (16.6 ± 7.9%) and CK19 (10.0 ± 4.3%) demonstrating the potential for epithelial transdifferentiation, whereas CK3 and CK19 were barely expressed in SHED when cultured alone. Expression of transcript levels of CK3 and CK19 were significantly upregulated when SHED were transwell cocultured or mixed cultured with HCE-T cells by 7, 14, and 21 days. CONCLUSIONS: We have demonstrated that SHED retain the potential for transdifferentiation to corneal epithelium-like cells by in vitro coculture with immortal corneal epithelium cells. Thus, exfoliated teeth may be an alternative tissue resource for providing stem cells for potential clinical applications in ocular surface regeneration.

The effects of acacia honey on in vitro corneal abrasion wound healing model.

BACKGROUND: Acacia honey (AH) has been proven to improve skin wound healing, but its therapeutic effects on corneal epithelium has not been elucidated to date. This study aimed to investigate the effects of AH on cultured corneal epithelial cells (CEC) on in vitro corneal abrasion wound healing model. Six New Zealand white rabbits' CEC were isolated and cultured until passage 1. Circular wound area was created onto a confluent monolayer CEC using a corneal trephine which mimicked corneal abrasion and treated with 0.025% AH supplemented in basal medium (BM) and complete cornea medium (CCM). Wound healing was measured as the percentage of wound closure by the migration of CEC on day 0, day 3 and day 6, post wound creation. The morphological changes of CEC were assessed via phase contrast microscopy. Gene and protein expressions of cytokeratin (CK3), fibronectin and cluster of differentiation 44 (CD44) in AH treated groups and control groups were determined by real-time PCR and immunocytochemistry, respectively. RESULTS: Cultured CEC exhibited similar morphology of polygonal shaped cells in all culture media. CEC cultured in AH-supplemented media showed higher percentage of wound closure compared to the controls. Gene expression of CK3 increased in AH-supplemented groups throughout the study. Fibronectin expression was increased at the initial stage while CD44 expression was increased at day 3, post wound creation. The protein expression of CEC cultured in all media was in accordance to their respective gene expressions. CONCLUSION: Supplementation of AH in BM and CCM media accelerates CEC wound closure of the in vitro corneal abrasion model by increasing the expression of genes and proteins associated with CEC wound healing.

Identification of presumed pathogenic KRT3 and KRT12 gene mutations associated with Meesmann corneal dystrophy.

PURPOSE: To report potentially pathogenic mutations in the keratin 3 (KRT3) and keratin 12 (KRT12) genes in two individuals with clinically diagnosed Meesmann corneal dystrophy (MECD). METHODS: Slit-lamp examination was performed on the probands and available family members to identify characteristic features of MECD. After informed consent was obtained, saliva samples were obtained as a source of genomic DNA, and screening of KRT3 and KRT12 was performed. Potentially pathogenic variants were screened for in 200 control chromosomes. PolyPhen-2, SIFT, and PANTHER were used to predict the functional impact of identified variants. Short tandem repeat genotyping was performed to confirm paternity. RESULTS: Slit-lamp examination of the first proband demonstrated bilateral, diffusely distributed, clear epithelial microcysts, consistent with MECD. Screening of KRT3 revealed a heterozygous missense variant in exon 1, c.250C>T (p.(Arg84Trp)), which has a minor allele frequency of 0.0076 and was not identified in 200 control chromosomes. In silico analysis with PolyPhen-2 and PANTHER predicted the variant to be damaging to protein function; however, SIFT analysis predicted tolerance of the variant. The second proband demonstrated bilateral, diffusely distributed epithelial opacities that appeared gray-white on direct illumination and translucent on retroillumination. Neither parent demonstrated corneal opacities. Screening of KRT12 revealed a novel heterozygous insertion/deletion variant in exon 6, c.1288_1293delinsAGCCCT (p.(Arg430_Arg431delinsSerPro)). This variant was not present in either of the proband's parents or in 200 control chromosomes and was predicted to be damaging by PolyPhen-2, PANTHER, and SIFT. Haplotype analysis confirmed paternity of the second proband, indicating that the variant arose de novo. CONCLUSIONS: We present a novel KRT12 mutation, representing the first de novo mutation and the first indel in KRT12 associated with MECD. In addition, we report a variant of uncertain significance in KRT3 in an individual with MECD. Although the potential pathogenicity of this variant is unknown, it is the first variant affecting the head domain of K3 to be reported in an individual with MECD and suggests that disease-causing variants associated with MECD may not be restricted to primary sequence alterations of either the helix-initiation or helix-termination motifs of K3 and K12.

Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane.

The cornea can be damaged by a variety of clinical disorders or chemical, mechanical, and thermal injuries. The objectives of this study were to induce bone marrow mesenchymal stem cells (BMSCs) to corneal lineage, to form a tissue engineered corneal substitute (TEC) using BMSCs, and to treat corneal surface defects in a limbal stem cell deficiency model. BMSCs were induced to corneal lineage using limbal medium for 10 days. Induced BMSCs demonstrated upregulation of corneal stem cell markers; ß1-integrin, C/EBPδ, ABCG2, and p63, increased protein expression of CK3 and p63 significantly compared with the uninduced ones. For TEC formation, passage 1 BMSCs were trypsinized and seeded on amniotic membrane in a transwell co-culture system and were grown in limbal medium. Limbal stem cell deficiency models were induced by alkaline injury, and the TEC was implanted for 8 weeks. Serial slit lamp evaluation revealed remarkable improvement in corneal regeneration in terms of corneal clarity and reduced vascularization. Histologic and optical coherence tomography analyses demonstrated comparable corneal thickness and achieved stratified epithelium with a compact stromal layer resembling that of normal cornea. CK3 and p63 were expressed in the newly regenerated cornea. In conclusion, BMSCs can be induced into corneal epithelial lineage, and these cells are viable for the formation of TEC, to be used for the reconstruction of the corneal surface in the limbal stem cell deficient model.

Autosomal-dominant Meesmann epithelial corneal dystrophy without an exon mutation in the keratin-3 or keratin-12 gene in a Chinese family.

Meesmann epithelial corneal dystrophy (MECD) is a dominantly inherited disorder, characterized by fragility of the anterior corneal epithelium and formation of intraepithelial microcysts. It has been described in a number of different ancestral groups. To date, all reported cases of MECD have been associated with either a single mutation in one exon of the keratin-3 gene (KRT3) or a single mutation in one of two exons of the keratin-12 gene (KRT12). Each mutation leads to a predicted amino acid change in the respective keratin-3 or keratin-12 proteins that combine to form the corneal-specific heterodimeric intermediate filament protein. This case report describes a four-generation Chinese kindred with typical autosomal-dominant MECD. Exon sequencing of KRT3 and KRT12 in six affected and eight unaffected individuals (including two spouses) did not detect any mutations or nucleotide sequence variants. This kindred demonstrates that single mis-sense mutations may be sufficient but are not required in all individuals with the MECD phenotype. It provides a unique opportunity to investigate further genomic and functional heterogeneity in MECD.

Comparison of culture media for ex vivo cultivation of limbal epithelial progenitor cells.

PURPOSE: To compare the effectiveness of three culture media for growth, proliferation, differentiation, and viability of ex vivo cultured limbal epithelial progenitor cells. METHODS: Limbal epithelial progenitor cell cultures were established from ten human corneal rims and grew on plastic wells in three culture media: supplemental hormonal epithelial medium (SHEM), keratinocyte serum-free medium (KSFM), and Epilife. The performance of culturing limbal epithelial progenitor cells in each medium was evaluated according to the following parameters: growth area of epithelial migration; immunocytochemistry for adenosine 5'-triphosphate-binding cassette member 2 (ABCG2), p63, Ki67, cytokeratin 3 (CK3), and vimentin (VMT) and real-time reverse transcription polymerase chain reaction (RT-PCR) for CK3, ABCG2, and p63, and cell viability using Hoechst staining. RESULTS: Limbal epithelial progenitor cells cultivated in SHEM showed a tendency to faster migration, compared to KSFM and Epilife. Immunocytochemical analysis showed that proliferated cells in the SHEM had lower expression for markers related to progenitor epithelial cells (ABCG2) and putative progenitor cells (p63), and a higher percentage of positive cells for differentiated epithelium (CK3) when compared to KSFM and Epilife. In PCR analysis, ABCG2 expression was statistically higher for Epilife compared to SHEM. Expression of p63 was statistically higher for Epilife compared to SHEM and KSFM. However, CK3 expression was statistically lower for KSFM compared to SHEM. CONCLUSIONS: Based on our findings, we concluded that cells cultured in KSFM and Epilife media presented a higher percentage of limbal epithelial progenitor cells, compared to SHEM.

Severe Meesmann's epithelial corneal dystrophy phenotype due to a missense mutation in the helix-initiation motif of keratin 12.

PURPOSE: To describe a severe phenotype of Meesmann's epithelial corneal dystrophy (MECD) and to determine the underlying molecular cause. METHODS: We identified a 30-member family affected by MECD and examined 11 of the 14 affected individuals. Excised corneal tissue from one affected individual was examined histologically. We used PCR and direct sequencing to identify mutation of the coding regions of the KRT3 and KRT12 genes. RESULTS: Cases had an unusually severe phenotype with large numbers of intraepithelial cysts present from infancy and they developed subepithelial fibrosis in the second to third decade. In some individuals, the cornea became superficially vascularized, a change accompanied by the loss of clinically obvious epithelial cysts. Visual loss from amblyopia or corneal opacity was common and four individuals were visually impaired (≤6/24 bilaterally) and one was blind (<6/60 bilaterally). In all affected family members, there was a heterozygous missense mutation c. 395T>C (p. L132P) in exon 1 of the KRT12 gene, which codes for the helix-initiation motif of the K12 polypeptide. This sequence change was not found in unaffected family members or in 100 unaffected controls. CONCLUSIONS: The Leu132Pro missense mutation is within the helix-initiation motif of the keratin and is predicted to result in a significant structural change of the K12 protein. The clinical effects are markedly more severe than the phenotype usually associated with the Arg135Thr mutation within this motif, most frequently seen in European patients with MECD.

Comparative analysis of tumor markers and evaluation of their predictive value in patients with colorectal cancer.

BACKGROUND: We evaluated the feasibility of CEA/CK20 mRNA and CEA/CA19-9 proteins as tumor markers for colorectal cancer by detecting tumor-specific mRNAs in circulating tumor cells and secreted tumor-specific proteins in the peripheral blood of colorectal cancer patients. PATIENTS AND METHODS: Peripheral blood was obtained from 23 healthy volunteers and 46 colorectal cancer patients on the day of initiation of adjuvant chemotherapy after surgery (stages I-III, n = 27) or on the first day of chemotherapy after diagnosis (stage IV, n = 19). Levels of CEA/CK20 mRNA in peripheral blood mononuclear cells (PBMCs) were determined with quantitative real-time reverse transcription polymerase chain reaction, and serum CEA/CA19-9 protein levels were determined by radioimmunoassay. RESULTS: The detection sensitivity of CK20 mRNA was approximately 1 tumor cell in 1 × 10(7) PBMCs, and that of CEA mRNA was approximately 1 tumor cell in 1 × 10(6) PBMCs. Patients with stage IV colorectal cancer had higher levels of CEA mRNA, CK20 mRNA, and serum CEA than patients at stages I-III. Peripheral blood CEA mRNA levels were predictive of overall survival, while serum protein levels of CEA and CA19-9 had no predictive value. CONCLUSION: Peripheral blood CEA mRNA is a useful marker of overall survival in colorectal cancer patients, that is sensitive and specific.

Pax-6 is expressed early in the differentiation of a corneal epithelial model system.

Pax-6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1-(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax-6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax-6-positive cells were also K3 keratin-positive, suggesting that Pax-6 is expressed in differentiating cells. Pax-6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT-qPCR. The raise in Pax-6 expression preceded for 9 h the increase in LDH-H and LDH-M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full-length mRNAs encoding for the two major Pax-6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax-6 mRNA was 2- to 2.5-fold more abundant than Pax-6(5a) mRNA. The ectopic expression of Pax-6 or Pax-6(5a) decreased proliferative ability leading to the formation of abortive, non-proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax-6. Collectively, results show that Pax-6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax-6 as the main driver of the differentiation of corneal epithelial cells.

Greater growth potential of p63-positive epithelial cell clusters maintained in human limbal epithelial sheets.

PURPOSE: To determine the growth potential of p63-positive cell clusters maintained in human limbal epithelial sheets. METHODS: Intact human limbal epithelial sheets were isolated from corneoscleral rims and cultured with or without being rendered into single cells by trypsinization on either plastic or 3T3 fibroblast feeder layers. Clonal growth on 3T3 fibroblast feeder layers was compared between monolayers in sheets or single cells and between areas with or without laser-microdissected, p63-enriched cell clusters. Immunostaining, immunoblot analysis, and reverse transcription-polymerase chain reaction of such differentiation markers as keratin (K)-3 and -12 and such progenitor markers as p63, ABCG2, and MDR-1 were also compared. RESULTS: Clusters of small p63-positive cells were enriched in limbal palisades of dispase-isolated epithelial sheets immediately or after brief cultivation on plastic in SHEM. Clonal growth of p63-rich cell clusters was higher than areas without clusters (P < 0.001). Clonal growth of epithelial monolayers derived from sheets was also higher than that derived from single cells (P < 0.01). Although expression of ABCG2 and MDR-1 transcripts was similar, cells from epithelial sheets expressed higher protein levels of p63 and lower protein levels of K3 and -12 than single cells, whether they were cultured on plastic or as 3T3 fibroblast feeder layers. CONCLUSIONS: Limbal palisades contain clusters of p63-rich progenitor cells. Maintenance of such cluster architecture during ex vivo expansion yields higher growth potential than being dispersed into single cells.

Genetics of Meesmann corneal dystrophy: a novel mutation in the keratin 3 gene in an asymptomatic family suggests genotype-phenotype correlation.

PURPOSE: Juvenile epithelial corneal dystrophy of Meesmann (MCD, OMIM 122100) is a dominantly inherited disorder characterized by fragility of the anterior corneal epithelium and intraepithelial microcyst formation. Although the disease is generally mild and affected individuals are often asymptomatic, some suffer from recurrent erosions leading to lacrimation, photophobia, and deterioration in visual acuity. MCD is caused by mutations in keratin 3 (KRT3) or keratin 12 (KRT12) genes, which encode cornea-specific cytoskeletal proteins. Seventeen mutations in KRT12 and two in KRT3 have been described so far. The purpose of this study was to investigate the genetic background of MCD in a Polish family. METHODS: We report on a three-generation family with MCD. Epithelial lesions characteristic for MCD were visualized with slit-lamp examination and confirmed by in vivo confocal microscopy. Using genomic DNA as a template, all coding regions of KRT3 and KRT12 were amplified and sequenced. Presence of the mutation was verified with restriction endonuclease digestion. RESULTS: In the proband, direct sequencing of the polymerase chain reaction (PCR) product from amplified coding regions of KRT3 and KRT12 revealed a novel 1493A>T heterozygous missense mutation in exon 7 of KRT3, which predicts the substitution of glutamic acid for valine at codon 498 (E498V). Using PCR-Restriction Fragment Length Polymorphism (RFLP) analysis, the mutation was demonstrated to segregate with the disease (four affected members, three non-affected) and to be absent in 100 controls from the Polish population, indicating that it is not a common polymorphism. CONCLUSIONS: Location of the E498V mutation emphasizes the functional relevance of the highly conserved boundary motifs at the COOH-terminus of the alpha-helical rod domain in keratin 3 (K3).