Publications (32)243.25 Total impact
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Article: Methods for characterization/manipulation of human corneal stem cells and their applications in regenerative medicine.
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ABSTRACT: Cell therapy is an emerging therapeutic strategy aimed at replacing or repairing severely damaged tissues with cultured cells. Specifically, ocular burns cause depletion of limbal stem cells, which leads to corneal opacification and visual loss. Corneal stem cells are segregated in the basal layer of the limbus, which is the transitional zone of the epithelium located between the cornea and the bulbar conjunctiva. Autologous cultured limbal epithelial cells can restore damaged corneas. We sought to establish a culture system that allows preservation of limbal stem cells and preparation of manageable epithelial sheets. We outline some quality criteria, which assure the clinical performance of keratinocyte culture: evaluation of the number of holoclones within a cultured epithelial graft, proportion of aborting colonies, and percentage of cells expressing high levels of ΔNp63α.Methods in molecular biology (Clifton, N.J.) 01/2012; 916:357-72. -
Article: Vision from the right stem.
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ABSTRACT: Cultures of limbal cells are a safe and effective treatment for the destruction of the human cornea owing to chemical burns. The essential feature of the graft is the presence of an adequate number of stem cells, which can be determined by the expression of the p63 transcription factor. Here, we will discuss the general principles defining the rigorous criteria for graftable limbal cultures in light of their clinical performances. Such criteria might prove relevant to the future therapeutic use of any cultured cell type.Trends in Molecular Medicine 11/2010; · 10.35 Impact Factor -
Article: Limbal stem-cell therapy and long-term corneal regeneration.
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ABSTRACT: Corneal renewal and repair are mediated by stem cells of the limbus, the narrow zone between the cornea and the bulbar conjunctiva. Ocular burns may destroy the limbus, causing limbal stem-cell deficiency. We investigated the long-term clinical results of cell therapy in patients with burn-related corneal destruction associated with limbal stem-cell deficiency, a highly disabling ocular disease. We used autologous limbal stem cells cultivated on fibrin to treat 112 patients with corneal damage, most of whom had burn-dependent limbal stem-cell deficiency. Clinical results were assessed by means of Kaplan-Meier, Kruskal-Wallis, and univariate and multivariate logistic-regression analyses. We also assessed the clinical outcome according to the percentage of holoclone-forming stem cells, detected as cells that stain intensely (p63-bright cells) in the cultures. Permanent restoration of a transparent, renewing corneal epithelium was attained in 76.6% of eyes. The failures occurred within the first year. Restored eyes remained stable over time, with up to 10 years of follow-up (mean, 2.91+/-1.99; median, 1.93). In post hoc analyses, success--that is, the generation of normal epithelium on donor stroma--was associated with the percentage of p63-bright holoclone-forming stem cells in culture. Cultures in which p63-bright cells constituted more than 3% of the total number of clonogenic cells were associated with successful transplantation in 78% of patients. In contrast, cultures in which such cells made up 3% or less of the total number of cells were associated with successful transplantation in only 11% of patients. Graft failure was also associated with the type of initial ocular damage and postoperative complications. Cultures of limbal stem cells represent a source of cells for transplantation in the treatment of destruction of the human cornea due to burns.New England Journal of Medicine 07/2010; 363(2):147-55. · 53.30 Impact Factor -
Article: Human embryonic stem cell-derived keratinocytes: how close to clinics?
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ABSTRACT: Recently in The Lancet, Guenou et al. (2009) demonstrate that human embryonic stem cells (hESCs) can differentiate into mature keratinocytes able to generate a pluristratified epithelium on immunodeficient mice. Their findings and the potential clinical use of hESC-derived keratinocytes will be discussed.Cell stem cell 01/2010; 6(1):8-9. · 23.56 Impact Factor -
Article: In vitro evidence of nerve growth factor effects on human conjunctival epithelial cell differentiation and mucin gene expression.
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ABSTRACT: Mucins released into the tear film are crucial to maintaining a healthy ocular surface. Alterations in goblet cell numbers and mucin secretion are observed in chronic ocular surface inflammatory diseases. Nerve growth factor (NGF) plays a crucial role in healing and inflammation of the ocular surface. The aim of this study was to evaluate in vitro the effect of NGF on conjunctival goblet cell differentiation and mucin production and secretion. Human conjunctival epithelial cells were exposed to increasing NGF concentrations (1 to 250 ng/mL) and analyzed to quantify cell growth (MTT/Ki67/BrdU), goblet cell differentiation (PAS/MUC5AC confocal staining), and mucin mRNA expression (real-time PCR). Secreted and cellular MUC5AC were also analyzed by sandwich-ELISA and FACS, respectively. To confirm the biological effects of NGF, the same evaluations were performed on primary cultures, and changes in markers of stemness (p63) and commitment (14-3-3 sigma) were also investigated. In cell cultures, NGF induced a dose-dependent increase of goblet cell numbers, MUC5AC production, storage, and release. Additionally, in primary cultures, NGF induced an increase of abortive colonies and 14-3-3 sigma protein, and a decrease of p63 mRNA and protein, suggesting a differentiating effect of NGF on human conjunctival epithelium. These findings show that NGF might play a role in the complex mechanism leading to conjunctival epithelium differentiation and mucin secretion. In addition to the known roles of NGF in promoting ocular surface healing and sensitivity, its effects on conjunctival goblet cells support a rationale to investigate the therapeutic effectiveness of NGF in dry eye disease.Investigative ophthalmology & visual science 05/2009; 50(10):4622-30. · 3.43 Impact Factor -
Article: Correction of laminin-5 deficiency in human epidermal stem cells by transcriptionally targeted lentiviral vectors.
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ABSTRACT: Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer-restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.Molecular Therapy (2008) 16 12, 1977-1985 doi:10.1038/mt.2008.204.Molecular Therapy 10/2008; 16(12):1977-85. · 6.87 Impact Factor -
Article: Towards therapeutic application of ocular stem cells.
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ABSTRACT: The first example of cell therapy using cultured stem cells dates back to 1981, when it was demonstrated that human epidermis could be grown in the laboratory and transplanted onto burnt patients to reconstitute a functional epidermis [Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 1979;76(11):5665-8; Banks-Schlegel S, Kehinde O, Green H. Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet 1981;1:75-8; Gallico 3rd GG, O'Connor NEMJ, Compton CC, Kehinde O, Green H. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984;311(7):448-51]. This was the onset of regenerative medicine, which is now being developed also in many other fields including ophthalmology. Emerging cell therapies for the restoration of sight have focused on two areas of the eye that are critical for visual function, the cornea and the retina. The relatively easy access of the cornea, the homogeneity of the cells forming the different layers of the corneal epithelium and the improvement of cell culture protocols are leading to considerable success in corneal epithelium restoration. Rebuilding the entire cornea is however still far from reality. The restoration of the retina has recently been achieved in different animal models of retinal degeneration using immature photoreceptors, and two other promising strategies have been demonstrated: transplantation of endothelial precursors to rescue retinal vessels and neurons, and transplantation of retinal pigmented epithelial cells to preserve vision over the long term. The relevance of these approaches will be discussed in function of the disease targeted.Seminars in Cell and Developmental Biology 01/2008; 18(6):805-18. · 6.65 Impact Factor -
Article: C/EBPdelta regulates cell cycle and self-renewal of human limbal stem cells.
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ABSTRACT: Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein delta (C/EBPdelta), Bmi1, and DeltaNp63alpha identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBPdelta and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBPdelta inhibits the growth of limbal colonies and increases the cell cycle length of primary limbal cells through the activity of p27(Kip1) and p57(Kip2). These effects are reversible; do not alter the limbal cell proliferative capacity; and are not due to apoptosis, senescence, or differentiation. C/EBPdelta, but not DeltaNp63alpha, indefinitely promotes holoclone self-renewal and prevents clonal evolution, suggesting that self-renewal and proliferation are distinct, albeit related, processes in limbal stem cells. C/EBPdelta is recruited to the chromatin of positively (p27(Kip1) and p57(Kip2)) and negatively (p16(INK4A) and involucrin) regulated gene loci, suggesting a direct role of this transcription factor in determining limbal stem cell identity.The Journal of Cell Biology 07/2007; 177(6):1037-49. · 10.26 Impact Factor -
Article: C/EBPδ regulates cell cycle and self-renewal of human limbal stem cells
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ABSTRACT: Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein δ (C/EBPδ), Bmi1, and ΔNp63α identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBPδ and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBPδ inhibits the growth of limbal colonies and increases the cell cycle length of primary limbal cells through the activity of p27Kip1 and p57Kip2. These effects are reversible; do not alter the limbal cell proliferative capacity; and are not due to apoptosis, senescence, or differentiation. C/EBPδ, but not ΔNp63α, indefinitely promotes holoclone self-renewal and prevents clonal evolution, suggesting that self-renewal and proliferation are distinct, albeit related, processes in limbal stem cells. C/EBPδ is recruited to the chromatin of positively (p27Kip1 and p57Kip2) and negatively (p16INK4A and involucrin) regulated gene loci, suggesting a direct role of this transcription factor in determining limbal stem cell identity.The Journal of Cell Biology 06/2007; 177(6):1037-1049. · 10.26 Impact Factor -
Article: Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts in response to wound healing.
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ABSTRACT: To characterize the expression of the visual system homeobox gene (VSX1) in human corneal keratocytes both in vitro and in vivo. The expression of VSX1 was evaluated through semiquantitative RT-PCR, immunofluorescence and in situ hybridization both in corneas (either freshly obtained or wounded) and in collagenase/hyaluronidase-isolated keratocytes grown in the absence or presence of serum to promote keratocyte-to-myofibroblast differentiation. Quiescent or resting keratocytes normally residing in the corneal stroma or cultured in vitro in the absence of serum did not express VSX1. In wounded corneas or when cultured in the presence of serum to mimic wound-healing responses, keratocytes underwent fibroblastic transformation (with appearance of alpha-SMA and disappearance of CD-34 and keratocan signals) and started expressing VSX1. The results show that VSX1 is expressed in vitro and in vivo during human corneal wound healing, a process in which differentiation of corneal keratocytes into myofibroblasts occurs. These data may help to elucidate the role of VSX1 in cornea physiology suggesting a potential involvement in cornea-related diseases such as keratoconus.Investigative Ophthalmology & Visual Science 01/2007; 47(12):5243-50. · 3.60 Impact Factor -
Article: Q-FIHC: quantification of fluorescence immunohistochemistry to analyse p63 isoforms and cell cycle phases in human limbal stem cells.
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ABSTRACT: Fluorescence microscopy has long been used for qualitative characterization of various parameters such as subcellular distribution of proteins, lipids, nucleic acids, and ions. However, quantification of these parameters is complicated by a variety of optical, biological, and physical factors. In the last decade, the progress achieved with powerful softwares and digital image processing systems has facilitated the development of fluorescence immunohistochemistry (FIHC) into a widely used quantitative assay (quantitative-FIHC or Q-FIHC). We describe here a rapid and sensitive Q-FIHC assay based on the use of a laser scanning confocal microscope and advanced image analysis softwares (Zeiss semi automatic LSM 510 and fully automatic Axiovision 4.4) for the detection and quantification of fluorescent intensity in human corneal tissues and cells obtained from small clinical samples. We have used this methodology to characterize and quantify the gene expression profile of p63 and its DeltaNalpha isoform, specific markers of human limbal stem cells. The validity of this method was evaluated through comparative studies with conventional approaches suggesting no significant differences and providing an alternative technique to traditional methods. Since Q-FIHC requires at least 20-fold less cells than traditional techniques, we have adopted it as the main quality control for our limbal cultures destined to clinical application.Microscopy Research and Technique 01/2007; 69(12):983-91. · 1.79 Impact Factor -
Article: Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.
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ABSTRACT: The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.Nature Medicine 01/2007; 12(12):1397-402. · 22.46 Impact Factor -
Article: Towards a gene therapy clinical trial for epidermolysis bullosa.
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ABSTRACT: Genetic mutations affecting the capacity of basal keratinocytes to adhere firmly to the underneath derma lead to severe, often lethal, blistering disorders of the skin known as Epidermolysis Bullosa (EB). About 400,000-500,000 people worldwide are affected and no definitive treatments have yet been developed. Gene therapy might represent an alternative therapeutic approach for these devastating inherited disorders. In the last 10 years pre-clinical studies have shown that human epidermal stem cells can be stably transduced using integrating vectors allowing long-term genetic correction of the adhesion defects affecting EB keratinocytes both in vitro and in vivo after transplantation onto immunodeficient animals. In addition tremendous progress have been achieved in the clinical applications of cultured keratinocytes (cell therapy) for the regeneration of the epidermis over full thickness wounds or the restoration of damaged corneal surfaces. The combination of (i) optimised culturing conditions not altering the epidermal stemness, (ii) gene transfer vectors able to target epidermal stem cells very efficiently and (iii) surgical procedures allowing the grafting of large skin areas have therefore led our group to submit the first phase I/II gene therapy clinical trial for Junctional Epidermolysis Bullosa.Reviews on Recent Clinical Trials 06/2006; 1(2):155-62. -
Article: Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides.
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ABSTRACT: An intronic point mutation was identified in the ocular albinism type 1 (OA1) gene (HUGO symbol, GPR143) in a family with the X-linked form of ocular albinism. Interestingly, the mutation creates a new acceptor splice site in intron 7 of the OA1 gene. In addition to low levels of normally spliced mRNA product of the OA1 gene, the patient samples contained also an aberrantly spliced mRNA with a 165 bp fragment of intron 7 (from position +750 to +914) inserted between exons 7 and 8. The abnormal transcript contained a premature stop codon and was unstable, as revealed by Northern blot analysis. We defined that mutation NC_000023.8:g.25288G>A generated a consensus binding motif for the splicing factor enhancer ASF/SF2, which most likely favored transcription of the aberrant mRNA. Furthermore, it activated a cryptic donor-splice site causing the inclusion between exons 7 and 8 of the 165 bp intronic fragment. Thus, the aberrant splicing is most likely explained by the generation of a de novo splicing enhancer motif. Finally, to rescue OA1 expression in the patient's melanocytes, we designed an antisense morpholino modified oligonucleotide complementary to the mutant sequence. The morpholino oligonucleotide (MO) was able to rescue OA1 expression and restore the OA1 protein level in the patient's melanocytes through skipping of the aberrant inclusion. The use of MO demonstrated that the lack of OA1 was caused by the generation of a new splice site. Furthermore, this technique will lead to new approaches to correct splice site mutations that cause human diseases.Human Mutation 05/2006; 27(5):420-6. · 5.69 Impact Factor -
Article: Gene therapy in combination with tissue engineering to treat epidermolysis bullosa.
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ABSTRACT: In the last 20 years epidermal stem cells have been extensively used for tissue regeneration of epidermis and other epithelial surfaces. The tremendous progress achieved has led to the development of protocols aimed at the correction of rare genetic disorders such as epidermolysis bullosa (EB), a severe, often lethal, blistering disorder of the skin. Approximately 400,000-500,000 people are affected worldwide and no definitive treatments have yet been developed. Gene therapy might represent an alternative therapeutic approach. This paper reviews the different strategies used to genetically modify keratinocytes from EB patients and addresses issues such as the use of in vivo or ex vivo approaches, how to target keratinocytes with stem cell properties in order to have long-term therapeutic gene expression, and which gene transfer agents should be used. The progress made has led the authors' group to submit a request for a Phase I/II ex vivo therapy clinical trial for patients with junctional EB.Expert opinion on biological therapy 05/2006; 6(4):367-78. · 3.22 Impact Factor -
Article: 811. Correction of Laminin-5 |[beta]|3 Chain Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors
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ABSTRACT: Molecular Therapy (2006) 13, S314|[ndash]|S315; doi: 10.1016/j.ymthe.2006.08.896 811. Correction of Laminin-5 |[beta]|3 Chain Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors Francesca Di Nunzio1, Giulietta Maruggi1, Stefano Ferrari2, Marcela del Rio3, Fernando Larcher3, Michele De Luca1 and Fulvio Mavilio11Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy2Veneto Eye Bank Foundation, Venice, Italy3Department of Molecular and Cell Biology, CIEMAT, Madrid, SpainMolecular Therapy 04/2006; · 6.87 Impact Factor -
Article: Regeneration of squamous epithelia from stem cells of cultured grafts.
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ABSTRACT: The only cultured cell types extensively used for tissue regeneration are the keratinocyte and the chondrocyte. Cultured autologous keratinocytes derived from the epidermis have been used for many years to produce grafts that regenerate an epidermis over a full-thickness wound, such as a third-degree burn. But there have been many failures of engraftment, and in the absence of criteria for the quality of the cultures, the causes of failure cannot be analyzed. It has become clear that the essential feature of the graft is the presence of an adequate number of stem cells. This article describes the criteria for estimating that number. Advances in graft preparation, combining better preservation of stem cells with ease of application of the graft, are also described. These improvements have been applied to cultures of ocular limbal cells, which contain the keratinocyte stem cells of the corneal epithelium. Cultures meeting the criteria of stem cell number have been grafted to 116 patients suffering from chemical destruction of the limbus. The procedure has been highly successful in the alleviation of suffering and the restoration of vision.Regenerative Medicine 02/2006; 1(1):45-57. · 3.72 Impact Factor -
Article: Isoforms of DeltaNp63 and the migration of ocular limbal cells in human corneal regeneration.
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ABSTRACT: The p63 gene generates transactivating and N-terminally truncated transcripts (DeltaNp63) initiated by different promoters. Alternative splicing gives rise to three different C termini, designated alpha, beta, and gamma. In the ocular epithelium, the corneal stem cells, which are segregated in the basal layer of the limbus, contain the alpha isoform but not beta or gamma. Holoclones derived from the limbus are rich in alpha, meroclones contain little, and paraclones contain none. In normal resting corneal epithelium, p63 of all isoforms is absent. Upon corneal wounding, cells originating from the limbus and containing alpha migrate progressively through the epithelium of the peripheral and central cornea. In the absence of an attached limbus, no alpha isoform appears in the corneal epithelium. When migrating cells containing the alpha isoform appear in the wounded corneal epithelium, they are confined to the basal layer, but the suprabasal cells, not only of the cornea but of the limbus as well, contain mRNA encoding beta and gamma. These data support the concept that the alpha isoform of p63 is necessary for the maintenance of the proliferative potential of limbal stem cells and their ability to migrate over the cornea. The beta and gamma isoforms, being suprabasal and virtually absent from the resting limbus, are not stem cell markers but are likely to play a role in epithelial differentiation specifically during the process of corneal regeneration.Proceedings of the National Academy of Sciences 08/2005; 102(27):9523-8. · 9.68 Impact Factor -
Article: Isoforms of ΔNp63 and the migration of ocular limbal cells in human corneal regeneration
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ABSTRACT: The p63 gene generates transactivating and N-terminally truncated transcripts (ΔNp63) initiated by different promoters. Alternative splicing gives rise to three different C termini, designated α, β, and γ. In the ocular epithelium, the corneal stem cells, which are segregated in the basal layer of the limbus, contain the α isoform but not β or γ. Holoclones derived from the limbus are rich in α, meroclones contain little, and paraclones contain none. In normal resting corneal epithelium, p63 of all isoforms is absent. Upon corneal wounding, cells originating from the limbus and containing α migrate progressively through the epithelium of the peripheral and central cornea. In the absence of an attached limbus, no α isoform appears in the corneal epithelium. When migrating cells containing the α isoform appear in the wounded corneal epithelium, they are confined to the basal layer, but the suprabasal cells, not only of the cornea but of the limbus as well, contain mRNA encoding β and γ. These data support the concept that the α isoform of p63 is necessary for the maintenance of the proliferative potential of limbal stem cells and their ability to migrate over the cornea. The β and γ isoforms, being suprabasal and virtually absent from the resting limbus, are not stem cell markers but are likely to play a role in epithelial differentiation specifically during the process of corneal regeneration.Proceedings of the National Academy of Sciences 07/2005; 102(27):9523-9528. · 9.68 Impact Factor -
Article: Separation of keratan-sulfate-derived disaccharides by high-performance liquid chromatography and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection.
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ABSTRACT: In this paper, we report a rapid, sensitive, and quantitative procedure to conduct disaccharide compositional analyses of keratan sulfates (KS) by means of high-performance liquid chromatography (HPLC) separation and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection of products generated by hydrolysis of this glycosaminoglycan with Bacillus sp. keratanase II or Escherichia freundii endo-beta-galactosidase. Following E. freundii endo-beta-galactosidase digestion of bovine corneal KS, the monosulfated disaccharide glcNAc6sbeta(1-->3)gal, accounting for approximately equals 95% nmol and 50% yield products, is produced. On the contrary, bovine corneal KS treated with endo-beta-N-acetylglucosaminidase (keratanase II) from Bacillus sp. generates two major products, the monosulfated disaccharide galbeta(1-->4)glcNAc6s ( approximately equals 50% nmol product) and the disulfated disaccharide gal6sbeta(1-->4)glcNAc6s ( approximately equals 40% nmol product) for over 90% nmol products. These disaccharides are separated and readily determined within 30 min by using a linear-gradient strong anion-exchange separation. A linear relationship was found for the two purified disaccharides over a wide range of concentrations, from approximately equals 108 pmol, 50 ng, to 2,160 pmol, 1,000 ng, for the disaccharide galbeta(1-->4)glcNAc6s, and from 92 pmol, 50 ng, to 1,840 pmol, 1,000 ng, for the disaccharide gal6sbeta(1-->4)glcNAc6s. HPLC analysis was applied to the quantitative and qualitative determination of KS produced by 3T3-J2 murine fibroblasts in the cell medium. The amount of KS was found to be 2.80+/-0.34 microg/ml/10(6) cells and composed of approximately equals 71% nmol of disaccharide galbeta(1-->4)glcNAc6s and 18% nmol of the disulfated disaccharide gal6sbeta(1-->4)glcNAc6s having approximately equals 1.20 sulfate groups/disaccharide. Our data illustrate that the HPLC procedure reported represents an improved approach for the quantitative and compositional microanalyses of KS, especially applicable to experimentation involving small amounts ( approximately 50 ng) of this glycosaminoglycan and in relation to its biological function and pathological importance.Analytical Biochemistry 07/2005; 342(2):200-5. · 3.00 Impact Factor
Top co-authors
Top Journals
Institutions
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2005–2012
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Università degli Studi di Modena e Reggio Emilia
- Center for Regenerative Medicine "Stefano Ferrari"
Modena, Emilia-Romagna, Italy
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2006
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Azienda ULSS numero 12 Veneziana
Quarto d’Altino, Veneto, Italy
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1999–2003
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Istituto Dermopatico dell'Immacolata
Roma, Latium, Italy
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2002
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Telethon Institute of Genetics and Medicine
Napoli, Campania, Italy
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1988
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National Institute for Research on Cancer
Genova, Liguria, Italy
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