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Graziella PELLEGRINI
Professore Ordinario
Dipartimento di Scienze della Vita sede Centro di Medicina Rigenerativa
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Pubblicazioni
2023
- Comparison between Cultivated Oral Mucosa and Ocular Surface Epithelia for COMET Patients Follow-Up
[Articolo su rivista]
Attico, Eustachio; Galaverni, Giulia; Torello, Andrea; Bianchi, Elisa; Bonacorsi, Susanna; Losi, Lorena; Manfredini, Rossella; Lambiase, Alessandro; Rama, Paolo; Pellegrini, Graziella
abstract
Total bilateral Limbal Stem Cell Deficiency is a pathologic condition of the ocular surface due to the loss of corneal stem cells. Cultivated oral mucosa epithelial transplantation (COMET) is the only autologous successful treatment for this pathology in clinical application, although abnormal peripheric corneal vascularization often occurs. Properly characterizing the regenerated ocular surface is needed for a reliable follow-up. So far, the univocal identification of transplanted oral mucosa has been challenging. Previously proposed markers were shown to be co-expressed by different ocular surface epithelia in a homeostatic or perturbated environment. In this study, we compared the transcriptome profile of human oral mucosa, limbal and conjunctival cultured holoclones, identifying Paired Like Homeodomain 2 (PITX2) as a new marker that univocally distinguishes the transplanted oral tissue from the other epithelia. We validated PITX2 at RNA and protein levels to investigate 10-year follow-up corneal samples derived from a COMET-treated aniridic patient. Moreover, we found novel angiogenesis-related factors that were differentially expressed in the three epithelia and instrumental in explaining the neovascularization in COMET-treated patients. These results will support the follow-up analysis of patients transplanted with oral mucosa and provide new tools to understand the regeneration mechanism of transplanted corneas.
2023
- Education for the translation of Advanced Therapy Medicinal Products
[Articolo su rivista]
Adamo, Davide; Attico, Eustachio; Pellegrini, Graziella
abstract
2023
- GSK-3 inhibition reverts mesenchymal transition in primary human corneal endothelial cells
[Articolo su rivista]
Maurizi, E.; Merra, A.; Macaluso, C.; Schiroli, D.; Pellegrini, G.
abstract
Human corneal endothelial cells are organized in a tight mosaic of hexagonal cells and serve a critical function in maintaining corneal hydration and clear vision. Regeneration of the corneal endothelial tissue is hampered by its poor proliferative capacity, which is partially retrieved in vitro, albeit only for a limited number of passages before the cells undergo mesenchymal transition (EnMT). Although different culture conditions have been proposed in order to delay this process and prolong the number of cell passages, EnMT has still not been fully understood and successfully counteracted. In this perspective, we identified herein a single GSK-3 inhibitor, CHIR99021, able to revert and avoid EnMT in primary human corneal endothelial cells (HCEnCs) from old donors until late passages in vitro (P8), as shown from cell morphology analysis (circularity). In accordance, CHIR99021 reduced expression of α-SMA, an EnMT marker, while restored endothelial markers such as ZO-1, Na+/K+ ATPase and N-cadherin, without increasing cell proliferation. A further analysis on RNA expression confirmed that CHIR99021 induced downregulation of EnMT markers (α-SMA and CD44), upregulation of the proliferation repressor p21 and revealed novel insights into the β-catenin and TGFβ pathways intersections in HCEnCs. The use of CHIR99021 sheds light on the mechanisms involved in EnMT, providing a substantial advantage in maintaining primary HCEnCs in culture until late passages, while preserving the correct morphology and phenotype. Altogether, these results bring crucial advancements towards the improvement of the corneal endothelial cells based therapy.
2023
- Lessons learnt, and still to learn, in first in human stem cell trials
[Articolo su rivista]
Barker, R. A.; Carpenter, M.; Jamieson, C. H. M.; Murry, C. E.; Pellegrini, G.; Rao, R. C.; Song, J.
abstract
Developing cellular therapies is not straightforward. This Perspective summarizes the experience of a group of academic stem cell investigators working in different clinical areas and aims to share insight into what we wished we knew before starting. These include (1) choosing the stem cell line and assessing the genome of both the starting and final product, (2) familiarity with GMP manufacturing, reagent validation, and supply chain management, (3) product delivery issues and the additional regulatory challenges, (4) the relationship between clinical trial design and preclinical studies, and (5) the market approval requirements, pathways, and partnerships needed.
2023
- Validation of airway porcine epithelial cells as an alternative to human in vitro preclinical studies
[Articolo su rivista]
Genna, V. G.; Adamo, D.; Galaverni, G.; Lepore, F.; Boraldi, F.; Quaglino, D.; Lococo, F.; Pellegrini, G.
abstract
Animal models are currently used in several fields of biomedical research as useful alternatives to human-based studies. However, the obtained results do not always effectively translate into clinical applications, due to interspecies anatomical and physiological differences. Detailed comparability studies are therefore required to verify whether the selected animal species could be a representative model for the disease or for cellular process under investigation. This has proven to be fundamental to obtaining reliable data from preclinical studies. Among the different species, swine is deemed an excellent animal model in many fields of biological research, and has been largely used in respiratory medicine, considering the high homology between human and swine airways. In the context of in vitro studies, the validation of porcine airway epithelial cells as an alternative to human epithelial cells is crucial. In this paper, porcine and human tracheal and bronchial epithelial cells are compared in terms of in vivo tissue architecture and in vitro cell behaviour under standard and airlifted conditions, analyzing the regenerative, proliferative and differentiative potentials of these cells. We report multiple analogies between the two species, validating the employment of porcine airway epithelial cells for most in vitro preclinical studies, although with some limitations due to species-related divergences.
2022
- Fluctuations in Corneal Endothelial LAP2 Expression Levels Correlate with Passage Dependent Declines in Their Cell Proliferative Activity
[Articolo su rivista]
Maurizi, E.; Merra, A.; Schiroli, D.; Ghezzi, B.; Macaluso, C.; Pellegrini, G.
abstract
The corneal endothelium is the inner corneal mono‐layered epithelium, fundamental for preserving corneal hydration and transparency. However, molecular mechanisms that regulate corneal endothelial cells (CEnCs), in particular regarding their proliferative capacity, have been only partially elucidated. CEnCs are quiescent in vivo and they easily undergo endothelial to mesenchymal transition (EnMT) in vitro. This study aims to analyze CEnCs behavior and expression in vitro, either in sub‐confluent growing (S) or confluent (C) CEnCs cultures. Primary rabbit and human CEnCs were cultured and used for RT‐PCR, immunofluorescence or western blot analysis. These methods allowed identifying a novel molecular marker, LAP2, that is upregulated in S while downregulated in C human or rabbit CEnCs. Those results were observed for several subsequent passages in culture and this, together with the correlation between ki67 and LAP2 expression, suggested LAP2 as a novel possible indicator for culture ageing. Finally, treatment with FGF and TGFβ in rCEnCs highlighted how LAP2 can vary as the cells regulate their proliferative state. In conclusion, we have identified a novel marker for CEnCs, LAP2, that regulates its expression depending on the cells sub/confluent state and that correlates with CEnCs proliferation.
2022
- Genetic Disorders of the Extracellular Matrix: From Cell and Gene Therapy to Future Applications in Regenerative Medicine
[Articolo su rivista]
Chakravarti, Shukti; Enzo, Elena; Rocha Monteiro de Barros, Maithê; Maffezzoni, Maria Benedetta Rizzarda; Pellegrini, Graziella
abstract
Metazoans have evolved to produce various types of extracellular matrix (ECM) that provide structural support, cell adhesion, cell-cell communication, and regulated exposure to external cues. Epithelial cells produce and adhere to a specialized sheet-like ECM, the basement membrane, that is critical for cellular homeostasis and tissue integrity. Mesenchymal cells, such as chondrocytes in cartilaginous tissues and keratocytes in the corneal stroma, produce a pericellular matrix that presents optimal levels of growth factors, cytokines, chemokines, and nutrients to the cell and regulates mechanosensory signals through specific cytoskeletal and cell surface receptor interactions. Here, we discuss laminins, collagen types IV and VII, and perlecan, which are major components of these two types of ECM. We examinegenetic defects in these components that cause basement membrane pathologies such as epidermolysis bullosa, Alport syndrome, rare pericellular matrix-related chondrodysplasias, and corneal keratoconus and discuss recent advances in cell and gene therapies being developed for some of these disorders.
2022
- Nanoneedles Induce Targeted siRNA Silencing of p16 in the Human Corneal Endothelium
[Articolo su rivista]
Maurizi, E.; Martella, D. A.; Schiroli, D.; Merra, A.; Mustfa, S. A.; Pellegrini, G.; Macaluso, C.; Chiappini, C.
abstract
Nanoneedles can target nucleic acid transfection to primary cells at tissue interfaces with high efficiency and minimal perturbation. The corneal endothelium is an ideal target for nanoneedle-mediated RNA interference therapy aimed at enhancing its proliferative capacity, necessary for tissue regeneration. This work develops a strategy for siRNA nanoninjection to the human corneal endothelium. Nanoneedles can deliver p16-targeting siRNA to primary human corneal endothelial cells in vitro without toxicity. The nanoinjection of siRNA induces p16 silencing and increases cell proliferation, as monitored by ki67 expression. Furthermore, siRNA nanoinjection targeting the human corneal endothelium is nontoxic ex vivo, and silences p16 in transfected cells. These data indicate that nanoinjection can support targeted RNA interference therapy for the treatment of endothelial corneal dysfunction.
2022
- Nanoneedles for targeted {siRNA} silencing of p16 in the Human Corneal Endothelium
[Articolo su rivista]
Maurizi, Eleonora; Alessandro Martella, Davide; Schiroli, Davide; Merra, Alessia; Ahmad Mustfa, Salman; Pellegrini, Graziella; Macaluso, Claudio; Chiappini, Ciro
abstract
2022
- SOX2 Is a Univocal Marker for Human Oral Mucosa Epithelium Useful in Post-COMET Patient Characterization
[Articolo su rivista]
Attico, Eustachio; Galaverni, Giulia; Bianchi, Elisa; Losi, Lorena; Manfredini, Rossella; Lambiase, Alessandro; Rama, Paolo; Pellegrini, Graziella
abstract
Total bilateral Limbal Stem Cells Deficiency is a pathologic condition of the ocular surface due to loss or impairment of corneal stem cell function, altering homeostasis of the corneal epithelium. Cultivated Oral Mucosa Epithelial Transplantation (COMET) is the only autologous treatment for this pathology. During the follow-up, a proper characterization of the transplanted oral mucosa on the ocular surface supports understanding the regenerative process. The previously proposed markers for oral mucosa identification (e.g., keratins 3 and 13) are co-expressed by corneal and conjunctival epithelia. Here, we propose a new specific marker to distinguish human oral mucosa from the epithelia of the ocular surface. We compared the transcriptome of holoclones (stem cells) from the human oral mucosa, limbal and conjunctival cultures by microarray assay. High expression of SOX2 identified the oral mucosa vs. cornea and conjunctiva, while PAX6 was highly expressed in corneal and conjunctival epithelia. The transcripts were validated by qPCR, and immunological methods identified the related proteins. Finally, the proposed markers were used to analyze a 10-year follow-up aniridic patient treated by COMET. These findings will support the follow-up analysis of COMET treated patients and help to shed light on the mechanism of corneal repair and regeneration.
2022
- The Growing Medical Need for Tracheal Replacement: Reconstructive Strategies Should Overcome Their Limits
[Articolo su rivista]
Adamo, D.; Galaverni, G.; Genna, V. G.; Lococo, F.; Pellegrini, G.
abstract
Breathing, being predominantly an automatic action, is often taken for granted. However, respiratory diseases affect millions of people globally, emerging as one of the major causes of disability and death overall. Among the respiratory dysfunctions, tracheal alterations have always represented a primary challenge for clinicians, biologists, and engineers. Indeed, in the case of wide structural alterations involving more than 50% of the tracheal length in adults or 30% in children, the available medical treatments are ineffective or inapplicable. So far, a plethora of reconstructive approaches have been proposed and clinically applied to face this growing, unmet medical need. Unfortunately, none of them has become a well-established and routinely applied clinical procedure to date. This review summarizes the main clinical reconstructive attempts and classifies them as non-tissue engineering and tissue engineering strategies. The analysis of the achievements and the main difficulties that still hinder this field, together with the evaluation of the forefront preclinical experiences in tracheal repair/replacement, is functional to promote a safer and more effective clinical translation in the near future.
2022
- The cell as a tool to understand and repair urethra
[Capitolo/Saggio]
Sceberras, Virginia; Maria Magrelli, Federica; Adamo, Davide; Maurizi, Eleonora; Attico, Eustachio; Giuseppe Genna, Vincenzo; Lazzeri, Massimo; Barbagli, Guido; Pellegrini, Graziella
abstract
2021
- Clinical Studies of COMET for Total LSCD: a Review of the Methods and Molecular Markers for Follow-Up Characterizations
[Articolo su rivista]
Attico, Eustachio; Galaverni, Giulia; Pellegrini, Graziella
abstract
Purpose of Review
This review outlines the main features of the clinical trials where cultivated oral mucosa epithelial cell transplantation (COMET) was performed, aiming to underscore a link between the clinical outcome and the expression of specific markers during the follow-up of patients, characteristic for a defined epithelium (cornea, oral mucosa, or conjunctiva) or related to vascularization.
Recent Findings
Currently, little is known about the reasons underlying the success or failure of COMET. To address this issue, we focused on tissue characterization at the molecular level, highlighting the findings concerning angiogenesis.
Summary
There are several discrepancies in the outcomes of COMET clinical trials. While some corneal/conjunctival markers can be considered reliable for understanding the biological mechanisms that drive corneal repair after transplants, a unique marker specifically expressed in the oral mucosa and an accurate study of the vascularization processes are currently missing. Together, these insights will help forecast successes and failures of these technologies.
2021
- Regenerative Medicine of Epithelia: Lessons From the Past and Future Goals
[Articolo su rivista]
Maurizi, Eleonora; Adamo, Davide; Magrelli, FEDERICA MARIA; Galaverni, Giulia; Attico, Eustachio; Merra, Alessia; Maffezzoni, MARIA BENEDETTA RIZZARDA; Losi, Lorena; Genna, VINCENZO GIUSEPPE; Sceberras, Virginia; Pellegrini, Graziella
abstract
This article explores examples of successful and unsuccessful regenerative medicine on human epithelia. To evaluate the applications of the first regenerated tissues, the analysis of the past successes and failures addresses some pending issues and lay the groundwork for developing new therapies. Research should still be encouraged to fill the gap between pathologies, clinical applications and what regenerative medicine can attain with current knowledge.
2020
- A fine-tuned β-catenin regulation during proliferation of corneal endothelial cells revealed using proteomics analysis
[Articolo su rivista]
Maurizi, E.; Schiroli, D.; Zini, R.; Limongelli, A.; Misto, R.; Macaluso, C.; Pellegrini, G.
abstract
Corneal endothelial (CE) dysfunction is the main indication for corneal transplantation, an invasive procedure with several limitations. Developing novel strategies to re-activate CE regenerative capacity is, therefore, of fundamental importance. This goal has proved to be challenging as corneal endothelial cells (CEnC) are blocked in the G0/G1 phase of the cell cycle in vivo and, albeit retaining proliferative capacity in vitro, this is further hindered by endothelial-to-mesenchymal transition. Herein we investigated the mechanisms regulating CEnC proliferation in vitro. Comparing the proteome of non-proliferating (in vivo—G0/G1) and proliferating (in vitro—G2/M) rabbit CEnC (rCEnC), 77 proteins, out of 3,328 identified, were differentially expressed in the two groups (p < 0.005). Literature and Gene Ontology analysis revealed β-catenin and transforming growth factor (TGF-β) pathways to be correlated with the identified proteins. Treatment of rCEnC with a β-catenin activator and inhibitor showed that β-catenin activation was necessary during rCEnC proliferation, but not sufficient for its induction. Furthermore, both pro-proliferative activity of basic fibroblast growth factor and anti-proliferative effects of TGF-β were regulated through β-catenin. Overall, these results provide novel insights into the molecular basis underlying the proliferation process that CEnC re-activate in vitro, consolidating the role of β-catenin and TGF-β.
2020
- Preclinical study for treatment of hypospadias by advanced therapy medicinal products
[Articolo su rivista]
Sceberras, V.; Attico, E.; Bianchi, E.; Galaverni, G.; Melonari, M.; Corradini, F.; Fantacci, M.; Ribbene, A.; Losi, L.; Balò, S.; Lazzeri, M.; Trombetta, C.; Rizzo, M.; Manfredini, R.; Barbagli, G.; Pellegrini, G.
abstract
Purpose
This paper explores the feasibility of a new therapy for the treatment of hypospadias patients. Hypospadias is a very common congenital malformation of male genitals, with very high rate of recurrences after surgery. The field of regenerative medicine, which offers innovative solutions for many pathologies, still does not offer reliable solution for this pathology. Here, we propose quality, safety, and clinical feasibility assessment for an oral mucosa advanced therapy medicinal product (ATMP) grown on a biocompatible scaffold for a clinical study on urethral reconstruction of hypospadias patients.
Methods
Urethral and oral mucosal epithelia from donor biopsies were cultivated between two fibrin layers, under clinical-grade conditions for cell and tissue characterization and comparison, aimed at tissue engineering. In addition, single-clone analyses were performed to analyze gene expression profiles of the two epithelia by microarray technology.
Results
Oral mucosa appeared suitable for urethral reconstruction. The resulting ATMP was proven to maintain stem cells and regenerative potency. The preclinical safety studies were performed on human tissues to assess abnormalities and tumorigenicity, and confirmed the safety of the ATMP. Finally, the patient selection and the clinical protocol for the upcoming clinical trial were defined.
Conclusions
Against this backdrop, in this paper, we are proposing a new reproducible and reliable ATMP for the treatment of hypospadias.
2020
- Surgery Versus ATMPs: An Example From Ophthalmology
[Articolo su rivista]
Magrelli, F. M.; Merra, A.; Pellegrini, G.
abstract
Advanced therapy medicinal products (ATMPs) are the new frontier of medicine. Advanced therapy medicinal products are set out to satisfy unmet medical needs and provide new innovative, cutting-edge therapies for serious or life-threatening diseases, thus providing new therapeutic options for people with few or no possibility of treatment. They are divided into four groups including gene therapy medicinal products, cell-based therapy medicinal products, tissue-engineered products, and combined ATMPs, which in Europe refer to products that incorporate one or more medical devices with any of the previously mentioned ATMPs as part of the advanced medicine product (AIFA, 2017; Ten Ham et al., 2018). Advanced therapy medicinal products can potentially have long-term benefits, thus bringing a long-lasting positive impact on patient health. Advanced therapy medicinal product therapies are often administered just once or twice, which gives patients the possibility to heal quickly compared to traditional therapies. They also provide a long-term saving opportunity, both in terms of costs of treatments and procedures that are no longer necessary and in terms of quality of life and productivity. The resolution of the patient’s illness has a monetary impact on the patient, the patient’s caretakers, and especially on the society (Alliance for Regenerative Medicine, 2019). The aim of this paper was to provide an overview on the use of ATMPs approved in Europe, with a focus on blindness and visual impairment and the related economic burden. In this case study, the effective cost of a blind patient in different European countries was compared after treatment with ATMPs or traditional therapies, focusing on visual impairment caused by corneal opacity. Our evaluation includes an overview of the global economic impact of the two types of therapies on the society. We estimated direct healthcare costs, direct non-healthcare costs, and labor productivity losses, to include costs on healthcare, services, patients, their families and for the society in general. We could conclude that the costs of the two therapeutic approaches are comparable.
2019
- Advances in stem cell research and therapeutic development
[Articolo su rivista]
De Luca, M.; Aiuti, A.; Cossu, G.; Parmar, M.; Pellegrini, G.; Robey, P. G.
abstract
Despite many reports of putative stem-cell-based treatments in genetic and degenerative disorders or severe injuries, the number of proven stem cell therapies has remained small. In this Review, we survey advances in stem cell research and describe the cell types that are currently being used in the clinic or are close to clinical trials. Finally, we analyse the scientific rationale, experimental approaches, caveats and results underpinning the clinical use of such stem cells.
2019
- Author Response to Letter “Other Causes of Limbal Stem Cell Deficiency”
[Articolo su rivista]
Deng, S. X.; Borderie, V.; Chan, C. C.; Dana, R.; Figueiredo, F. C.; Gomes, J. A. P.; Pellegrini, G.; Shimmura, S.; Kruse, F. E.
abstract
2019
- Bioreactor-manufactured cartilage grafts repair acute and chronic osteochondral defects in large animal studies
[Articolo su rivista]
Vukasovic, A.; Asnaghi, M. A.; Kostesic, P.; Quasnichka, H.; Cozzolino, C.; Pusic, M.; Hails, L.; Trainor, N.; Krause, C.; Figallo, E.; Filardo, G.; Kon, E.; Wixmerten, A.; Maticic, D.; Pellegrini, G.; Kafienah, W.; Hudetz, D.; Smith, T.; Martin, I.; Ivkovic, A.; Wendt, D.
abstract
Objectives: Bioreactor-based production systems have the potential to overcome limitations associated with conventional tissue engineering manufacturing methods, facilitating regulatory compliant and cost-effective production of engineered grafts for widespread clinical use. In this work, we established a bioreactor-based manufacturing system for the production of cartilage grafts. Materials & Methods: All bioprocesses, from cartilage biopsy digestion through the generation of engineered grafts, were performed in our bioreactor-based manufacturing system. All bioreactor technologies and cartilage tissue engineering bioprocesses were transferred to an independent GMP facility, where engineered grafts were manufactured for two large animal studies. Results: The results of these studies demonstrate the safety and feasibility of the bioreactor-based manufacturing approach. Moreover, grafts produced in the manufacturing system were first shown to accelerate the repair of acute osteochondral defects, compared to cell-free scaffold implants. We then demonstrated that grafts produced in the system also facilitated faster repair in a more clinically relevant chronic defect model. Our data also suggested that bioreactor-manufactured grafts may result in a more robust repair in the longer term. Conclusion: By demonstrating the safety and efficacy of bioreactor-generated grafts in two large animal models, this work represents a pivotal step towards implementing the bioreactor-based manufacturing system for the production of human cartilage grafts for clinical applications. Read the Editorial for this article on doi:10.1111/cpr.12625.
2019
- Global consensus on definition, classification, diagnosis, and staging of limbal stem cell deficiency
[Articolo su rivista]
Deng, S. X.; Borderie, V.; Chan, C. C.; Dana, R.; Figueiredo, F. C.; Gomes, J. A. P.; Pellegrini, G.; Shimmura, S.; Kruse, F. E.
abstract
Purpose: Despite extensive knowledge gained over the last 3 decades regarding limbal stem cell deficiency (LSCD), the disease is not clearly defined, and there is lack of agreement on the diagnostic criteria, staging, and classification system among treating physicians and research scientists working on this field. There is therefore an unmet need to obtain global consensus on the definition, classification, diagnosis, and staging of LSCD. Methods: A Limbal Stem Cell Working Group was first established by The Cornea Society in 2012. The Working Group was divided into subcommittees. Four face-to-face meetings, frequent email discussions, and teleconferences were conducted since then to obtain agreement on a strategic plan and methodology from all participants after a comprehensive literature search, and final agreement was reached on the definition, classification, diagnosis, and staging of LSCD. A writing group was formed to draft the current manuscript, which has been extensively revised to reflect the consensus of the Working Group. Results: A consensus was reached on the definition, classification, diagnosis, and staging of LSCD. The clinical presentation and diagnostic criteria of LSCD were clarified, and a staging system of LSCD based on clinical presentation was established. Conclusions: This global consensus provides a comprehensive framework for the definition, classification, diagnosis, and staging of LSCD. The newly established criteria will aid in the correct diagnosis and formulation of an appropriate treatment for different stages of LSCD, which will facilitate a better understanding of the condition and help with clinical management, research, and clinical trials in this area.
2019
- Laminin 332-Dependent YAP Dysregulation Depletes Epidermal Stem Cells in Junctional Epidermolysis Bullosa
[Articolo su rivista]
De Rosa, L.; Secone Seconetti, A.; De Santis, G.; Pellacani, G.; Hirsch, T.; Rothoeft, T.; Teig, N.; Pellegrini, G.; Bauer, J. W.; De Luca, M.
abstract
Laminin 332-deficient junctional epidermolysis bullosa (JEB) is a severe genetic skin disease. JEB is marked by epidermal stem cell depletion, the origin of which is unknown. We show that dysregulation of the YAP and TAZ pathway underpins such stem cell depletion. Laminin 332-mediated YAP activity sustains human epidermal stem cells, detected as holoclones. Ablation of YAP selectively depletes holoclones, while enforced YAP blocks conversion of stem cells into progenitors and indefinitely extends the keratinocyte lifespan. YAP is dramatically decreased in JEB keratinocytes, which contain only phosphorylated, inactive YAP. In normal keratinocytes, laminin 332 and alpha 6 beta 4 ablation abolish YAP activity and recapitulate the JEB phenotype. In JEB keratinocytes, laminin 332-gene therapy rescues YAP activity and epidermal stem cells in vitro and in vivo. In JEB cells, enforced YAP recapitulates laminin 332-gene therapy, thus uncoupling adhesion from proliferation in epidermal stem cells. This work has important clinical implication for ex vivo gene therapy of JEB.
2019
- New Drugs?
[Articolo su rivista]
Pellegrini, Graziella
abstract
2019
- Retinoic acid/calcite micro-carriers inserted in fibrin scaffolds modulate neuronal cell differentiation
[Articolo su rivista]
Barbalinardo, M.; Di Giosia, M.; Polishchuk, I.; Magnabosco, G.; Fermani, S.; Biscarini, F.; Calvaresi, M.; Zerbetto, F.; Pellegrini, G.; Falini, G.; Pokroy, B.; Valle, F.
abstract
The controlled release of cell differentiating agents is crucial in many aspects of regenerative medicine. Here we propose the use of hybrid calcite single crystals as micro-carriers for the controlled and localized release of retinoic acid, which is entrapped within the crystalline lattice. The release of retinoic acid occurs only in the proximity of stem cells, upon dissolution of the calcite hybrid crystals that are dispersed in the fibrin scaffold. These hybrid crystals provide a sustained dosage of the entrapped agent. The environment provided by this composite scaffold enables differentiation towards neuronal cells that form a three-dimensional neuronal network.
2019
- Stem Cells and Ocular Regeneration.
[Capitolo/Saggio]
Attico, E.; Chiavelli, Chiara; Sceberras, V.; Fantacci, M.; Melonari, M.; Pellegrini, Graziella
abstract
The surface of the eye is a peculiar and extraordinary area, since the functions of all regions of the ocular surface system (OSS) are closely integrated forming a unique functional unit composed of different tissues and organs.
Anatomically the ocular surface is composed principally by cornea and conjunctiva, but it also includes the lacrimal gland and the lacrimal drainage system for the tear film homeostasis, and the eyelids. Additional components as Meibomian glands, eyelashes and eyebrows complete the picture, contributing to the outermost lipid layer and to particle clearance (Fig. 1). Cornea and conjunc- tiva are generally covered by a complex liquid known as the tear film, providing a continuous moist environment connecting all areas at the front of the eye ball and protecting them from pathogens and small solid particles. This interconnected system provides the necessary conditions for the maintenance of functions needed for the vision process, as the final goal of eye function.
The dysfunction of this apparatus could lead to several pathologies, such as corneal opacity or dry eye diseases (DED).
Each component plays an important role for ocular surface maintenance, therefore some recent findings in stem cell biology aimed at the regeneration of different components of the ocular surface by tissue engineering, are examined in the following paragraphs.
2018
- Approaches for Effective Clinical Application of Stem Cell Transplantation
[Articolo su rivista]
Attico, Eustachio; Sceberras, VIRGINIA SIDONIA; Pellegrini, G.
abstract
Purpose of Review:
This review highlights problems related to translation of advanced therapy medicinal products (ATMPs) from bench to bedsite. Regenerative medicine within the current regulatory frame reveals common hitches in the course of development, translation, and clinical application. This paper suggests outlining a path from the few examples of successfully approved vs unsuccessful advanced therapies.
Recent Findings:
In the multitude of ongoing studies, few of them achieved positive results with a final treatment available to patients; this result was possible due to multidisciplinary teams working together from the beginning of the development and during the hard route to standardization and clinical application.
Summary:
The root of success of an advanced therapy requires not only the inescapable scientific and biological knowledge but also requires several contributions as regulatory, ethical, medical, and bio-engineering expertise, from the real beginning. A strong scientific rationale and an integrated network of expertises would contribute to a successful investment of available resources in advanced therapy medicinal products and to a greater confidence in future medicine.
2018
- Erratum: Regenerating Eye Tissues to Preserve and Restore Vision (Cell Stem Cell (2018) 22(6) (834–849), (S1934590918302315) (10.1016/j.stem.2018.05.013))
[Articolo su rivista]
Stern, J. H.; Tian, Y.; Funderburgh, J.; Pellegrini, G.; Zhang, K.; Goldberg, J. L.; Ali, R. R.; Young, M.; Xie, Y.; Temple, S.
abstract
(Cell Stem Cell 22, 834–849; June 1, 2018) The original labeling in Figure 1 incorrectly indicated that the regenerative cells in the postnatal lens epithelium are solely located in the equatorial region. Lineage studies indicate that these regenerative lens epithelial cells, identified in the text as stem/progenitor cells, are located widely in the anterior epithelium but preferentially proliferate in the equatorial region. The labeling has been changed to reflect what is known about the predominant location of this dividing population. We apologize for any confusion caused to our colleagues in the community.
2018
- Living with Keratinocytes
[Articolo su rivista]
Pellegrini, Graziella; De Luca, Michele
abstract
A feature distinguishing human hematopoietic and epithelial stem cells from other equally fascinating stem cells is perhaps their easier translation into a clinical setting. We have devoted nearly our entire scientific career in trying to turn our understanding of epithelial stem cell biology into something that could help people suffering from virtually untreatable diseases of squamous epithelia. We have done that as a team, together with our numerous students, postdocs, technicians and valuable collaborators, clinicians, regulators, and, lately, industrial partners. We had rewarding successes and burning failures, but we always did our best. This award, given by friends and colleagues deserving it more than us, has been the most important recognition of our work. Below, we summarize our story.
2018
- Navigating Market Authorization: The Path Holoclar Took to Become the First Stem Cell Product Approved in the European Union
[Articolo su rivista]
Pellegrini, Graziella; Ardigò, Diego; Milazzo, Giovanni; Iotti, Giorgio; Guatelli, Paolo; Pelosi, Danilo; De Luca, Michele
abstract
Gene therapy, cell therapy, and tissue engineering have the potential to revolutionize the treatment of disease and injury. Attaining marketing authorization for such advanced therapy medicinal products (ATMPs) requires a rigorous scientific evaluation by the European Medicines Agencyâauthorization is only granted if the product can fulfil stringent requirements for quality, safety, and efficacy. However, many ATMPs are being provided to patients under alternative means, such as âhospital exemptionâ schemes. Holoclar (ex vivo expanded autologous human corneal epithelial cells containing stem cells), a novel treatment for eye burns, is one of the few ATMPs to have been granted marketing authorization and is the first containing stem cells. This review highlights the differences in standards between an authorized and unauthorized medicinal product, and specifically discusses how the manufacture of Holoclar had to be updated to achieve authorization. The result is that patients will have access to a therapy that is manufactured to high commercial standards, and is supported by robust clinical safety and efficacy data. Stem Cells Translational Medicine 2018;7:146â154.
2018
- Regenerating Eye Tissues to Preserve and Restore Vision
[Articolo su rivista]
Stern, J. H.; Tian, Y.; Funderburgh, J.; Pellegrini, G.; Zhang, K.; Goldberg, J. L.; Ali, R. R.; Young, M.; Xie, Y.; Temple, S.
abstract
Ocular regenerative therapies are on track to revolutionize treatment of numerous blinding disorders, including corneal disease, cataract, glaucoma, retinitis pigmentosa, and age-related macular degeneration. A variety of transplantable products, delivered as cell suspensions or as preformed 3D structures combining cells and natural or artificial substrates, are in the pipeline. Here we review the status of clinical and preclinical studies for stem cell-based repair, covering key eye tissues from front to back, from cornea to retina, and including bioengineering approaches that advance cell product manufacturing. While recognizing the challenges, we look forward to a deep portfolio of sight-restoring, stem cell-based medicine. Video Abstract:[Figure presented] Stern et al. review the status of clinical and preclinical studies for stem cell-based repair, covering key eye tissues from front to back, from cornea to retina, and including bioengineering approaches that advance cell product manufacturing.
2018
- Tackling Ethical Challenges of Premature Delivery of Stem Cell-Based Therapies: ISSCR 2018 Annual Meeting Focus Session Report
[Articolo su rivista]
Sugarman, J.; Barker, R. A.; Kerridge, I.; Lysaght, T.; Pellegrini, G.; Sipp, D.; Tanner, C.
abstract
Clinical uses of unproven stem cell-based interventions abound, yet many patients may be harmed by receiving them, raising complex ethical, economic, and societal concerns. Regulators, scientists, clinicians, professional societies, and patient advocacy groups need to collaboratively articulate expectations related to the proper development and delivery of stem cell-based therapies. Clinical uses of unproven stem cell-based interventions abound, yet many patients may be harmed by receiving them, raising complex ethical, economic, and societal concerns. Regulators, scientists, clinicians, professional societies, and patient advocacy groups need to collaboratively articulate expectations related to the proper development and delivery of stem cell-based therapies.
2017
- Closure of a Large Chronic Wound through Transplantation of Gene-Corrected Epidermal Stem Cells
[Articolo su rivista]
Bauer, Johann; Koller, Josef; Murauer, Eva; DE ROSA, Laura; Enzo, Elena; Carulli, Sonia; Bondanza, Sergio; Recchia, Alessandra; Muss, Wolfgang; Diem, Anja; Mayr, Elisabeth; Schlager, Pamina; Gratz, Iris; Pellegrini, Graziella; DE LUCA, Michele
abstract
Generalized junctional epidermolysis bullosa (JEB) is caused by mutations in LAMA3,LAMB3,or
LAMC2,which together encode laminin-332, a hetero-trimeric protein consisting ofa3,b3, andg2chain. In nonlethal generalized intermediate JEB, laminin-332 is highly reduced, and hemidesmosomes are rudimentary or completely absent, leading to blister formation within the lamina lucida of the basement membrane upon minor trauma. The resulting chronic skin wounds invariably develop recurrent infections and scarring, which greatly impair patients’ quality of life. We report on a patient in whom gene-corrected epidermal sheets were transplanted onto a large nonhealing epidermal ulceration following a good manufacturing practice protocol
2017
- Cultivated limbal epithelial transplantation
[Articolo su rivista]
Rama, Paolo; Ferrari, Giulio; Pellegrini, Graziella
abstract
PURPOSE OF REVIEW: To provide an updated literature review on the status of cultivated limbal (corneal) epithelial transplantation. Cultivated limbal stem-cell transplantation recently received regulatory approval. We provide a comprehensive overview of recent developments in the field. RECENT FINDINGS: The current article reviews and highlights recent developments in the field of cultivated limbal stem-cell transplantation as retrieved from a literature search for the last year. SUMMARY: The implications of clinical/research findings in terms of transplanted cell source and cultivation methods in limbal stem-cell transplantation are reviewed.
2017
- In vitro method for producing a flap of genetically modified cells on fibrin substrate
[Brevetto]
Pellegrini, G.; Alessandrini, A.; De Luca, M.
abstract
2017
- Regeneration of the entire human epidermis using transgenic stem cells
[Articolo su rivista]
Hirsch, Tobias; Rothoeft, Tobias; Teig, Norbert; Bauer, Johann W.; Pellegrini, Graziella; De Rosa, Laura; Scaglione, Davide; Reichelt, Julia; Klausegger, Alfred; Kneisz, Daniela; Romano, Oriana; SECONE SECONETTI, Alessia; Contin, Roberta; Enzo, Elena; Jurman, Irena; Carulli, Sonia; Jacobsen, Frank; Luecke, Thomas; Lehnhardt, Marcus; Fischer, Meike; Kueckelhaus, Maximilian; Quaglino, Daniela; Morgante, Michele; Bicciato, Silvio; Bondanza, Sergio; De Luca, Michele
abstract
Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, lifethreatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.
2017
- Rigenerazione della cornea. Dalla scoperta all'approvazione di un prodotto medicinale di terapia avanzata contenente cellule staminali in Europa
[Capitolo/Saggio]
Pellegrini, G; Lambiase, Andrea; Macaluso, C; Pocobelli, A; Vinciguerra, P; Cavallini, Gm; Ducange, Pietro; Rama, P
abstract
2016
- Comparative assessment of cultures from oral and urethral stem cells for urethral regeneration
[Articolo su rivista]
Corradini, F.; Zattoni, Michela; Barbagli, G.; Bianchi, Giampaolo; Giovanardi, M.; Serafini, Chiara; Genna, VINCENZO GIUSEPPE; Ribbene, A.; Balò, S.; Fidanza, Francesco Antonio; Lazzeri, M.; DE LUCA, Michele; Pellegrini, Graziella
abstract
Urethral reconstruction has received much attention in recent years, due to pathologies such as recurrence of urethral strictures after treatments. Various surgical techniques have been developed to obtain the best risk-benefit ratio, such as autologous grafts taken from the oral cavity. Tissue engineering and stem cells, growing tissue from a small biopsies, can further improve surgery, reducing invasiveness and morbidity. To determine whether urethra or other epithelia can be equally useful for urethra engineering, a comparison of clonogenic ability, proliferative potential and stem cell markers should be obtained. In this study, 19 biopsies from urethra, and 21 from oral mucosa were obtained from patients, during reconstructive surgery. Urethral and oral tissues were removed from the same donor, to develop primary cultures and cell characterization. The long term regenerative properties of both tissues was investigated in vitro by life span, clonal analysis and markers of different clonal types. Results revealed the same high proliferative potential for urethra and oral mucosa cultures, but maintenance of specific markers. Karyotype and growth factor dependence confirmed the normal phenotype of cultured cells. Clonal analysis of the proliferative compartment highlighted a very different proportion of stem and transient amplifying cells, characterised by dissimilar cell size profile and marker expression. In conclusion, both tissues can be cultured and preserve their stem cells in vitro. Few differences appeared in oral mucosa vs urethra, suggesting that they can be equally useful for tissue engineering of the urethral tract.
2016
- From discovery to approval of an advanced therapy medicinal product-containing stem cells, in the EU
[Articolo su rivista]
Pellegrini, Graziella; Lambiase, Alessandro; Macaluso, Claudio; Pocobelli, Augusto; Deng, Sophie; Cavallini, Gian Maria; Esteki, Roza; Rama, Paolo
abstract
In 1997, the human corneal epithelium was reconstructed in vitro and transplanted on patients. Later, it became a routine treatment, before regulations considered advanced therapy medicinal products and drugs on the same lines. Manufacturing, before and after good manufacturing practice setting, was established in different facilities and the clinical application in several hospitals. Advanced therapy medicinal products, including stem cells, are unique products with different challenges than other drugs: some uncertainties, in addition to benefit, cannot be avoided. This review will focus on all recent developments in the stem cell based corneal therapy.
2016
- Holoclar: first of its kind in more ways than one
[Articolo su rivista]
Milazzo, G; Ardigò, D; Toschi, M; Matuska, S; Rama, P; DE LUCA, Michele; Pellegrini, Graziella
abstract
This article describes the regulatory pathway that enabled the translation of academic research and clinical experience of an ex-vivo expanded autologous
stem cell-based treatment for limbal stem cell deficiency (LSCD) into a pharmaceutical product compliant with the European Union regulations of Advanced Therapy Medicinal Products (ATMP). Holoclar® was originally developed in Italy as a surgical procedure and used in more than 200 patients. Following the establishment of the EU ATMP Regulation EC 1394/2007, Holoclar development required that manufacturing was as per current Good Manufacturing Practice requirements and collection of retrospective clinical data was ICH-E6 and E3 compliant. Holoclar is an ex-vivo expanded autologous human corneal epithelial cells containing stem cells and is classified as “tissue engineered product”. Based on the evidence of quality and control of the manufacturing process, safety, efficacy and on a positive benefit–risk balance in 104 patients (72.1%) of 148 patients treated, Holoclar received conditional Marketing Approval in the EU. Holoclar is the first medicine approved in the EU for this rare eye condition that can result in blindness.
2016
- One-stage Penile Urethroplasty Using Oral Mucosal Graft and Glue
[Articolo su rivista]
Barbagli, G.; Pellegrini, G.; Corradini, F.; Montorsi, F.; Sansalone, S.; Butnaru, D.; Lazzeri, M.
abstract
Background Repair of penile urethral strictures is a challenging problem for which different techniques have been suggested. Objective To describe a new surgical technique for one-stage penile urethroplasty using an oral graft and glue, and to assess its safety and efficacy. Design, setting, and participants A retrospective review of medical records for patients who underwent one-stage penile urethroplasty using oral mucosa and glue from February 2013 to October 2014 was performed. Surgical procedure The penile urethra was opened and the urethral plate was incised to create a wide window within which the oral graft was pasted with glue. The urethra was sutured over the catheter. Outcome measurements and statistical analysis Clinical data were collected in a database. Intraoperative and postoperative complications and outcomes were assessed. A descriptive statistical analysis was performed. Results and limitations Fourteen patients were included in the study. Median operative time was 60 min. The median postoperative stay was 3 d. Three intraoperative and one postoperative complication occurred. In all patients, voiding cystourethrography 2 wk after surgery failed to show urethral fistula or sacculation. No patients complained of penile chordee or sexual dysfunction after surgery. Median follow-up was 16 mo. Among the 14 patients, 12 (85.7%) procedures were successful and two (14.3%) were failures. Study limitations include the small sample size and short follow-up. Conclusions An in vitro study and a one-stage reconstruction of penile urethral strictures with an oral mucosa graft and glue showed that the procedure is safe and efficient, but further studies including larger series of patients and longer follow-up are required. Patient summary We report on the repair of penile urethral stricture using one-stage urethroplasty with oral mucosa and glue. This new technique was safe and effective, with limited complications and satisfactory outcomes. We plan to increase the use of this technique in the future.
2015
- Advances in Gene/Cell Therapy in Epidermolysis Bullosa
[Articolo su rivista]
Murauer, Eva M; Koller, Ulrich; Pellegrini, Graziella; DE LUCA, Michele; Bauer, Johann W.
abstract
In the past few years, substantial preclinical and experimental advances have been made in the treatment of the severe monogenic skin blistering disease epidermolysis bullosa (EB). Promising approaches have been developed in the fields of protein and cell therapies, including allogeneic stem cell transplantation; in addition, the application of gene therapy approaches has become reality. The first ex vivo gene therapy for a junctional EB (JEB) patient was performed in Italy more than 8 years ago and was shown to be effective. We have now continued this approach for an Austrian JEB patient. Further, clinical trials for a gene therapy treatment of recessive dystrophic EB are currently under way in the United States and in Europe. In this review, we aim to point out that sustainable correction of autologous keratinocytes by stable genomic integration of a therapeutic gene represents a realistic option for patients with EB.
2014
- Chemical injury treated with autologous limbal epithelial stem cell transplantation and subconjunctival bevacizumab
[Articolo su rivista]
Cavallini, Gian Maria; Pellegrini, Graziella; Volante, Veronica; Ducange, Pietro; DE MARIA, Michele; Torlai, Giulio; Benatti, Caterina; Forlini, Matteo
abstract
Limbal stem cell (LSC) deficiency leads to corneal opacity due to a conjunctivalization of the corneal surface. LSC transplantation, which can be followed by corneal keratoplasty, is an effective procedure to restore corneal transparency; however, a common cause of failure of this procedure is neovascularization (NV).
2014
- Concise review: hurdles in a successful example of limbal stem cell-based regenerative medicine
[Articolo su rivista]
Pellegrini, Graziella; P., Rama; DI ROCCO, Antonio; Panaras, Athanasios; DE LUCA, Michele
abstract
Recent breakthroughs in regenerative medicine have generated enthusiasm and many efforts to explore new therapeutic potentials of both somatic and pluripotent stem cells. About 30 years passed since a discovery of a method of producing a great number of human epidermal keratinocytes by cultivation from a small skin biopsy, many possibilities are now envisaged for therapeutic application of different cultured cell types. The importance of stem cell content was proven for many tissues or organs in different pathologies. Ocular burns cause depletion of limbal stem cells, which lead to corneal opacification and visual loss. Most of available treatments are palliative and focused on the relief of the devastating clinical picture. This review is focused on recent developments cell based therapy of limbal stem cell deficiency. All findings can provide support for improvement and standardization of the cure for this disabling disease. Stem Cells 2013.
2014
- Corneal Bioengineering
[Capitolo/Saggio]
Corradini, Francesca; Zattoni, Michela; Rama, Paolo; DE LUCA, Michele; Pellegrini, Graziella
abstract
The cornea is the main structure of the ocular surface and enables the transmission of light entering the eye. The cornea is covered by a nonkeratinized stratified epithelium, which is renewed by stem cells located in the basal layer of the limbus. Injury and disease, such as chemical and thermal burns, can destroy the limbus leading to a limbal stem cell deficiency (LSCD) with consequent visual loss. In 1997, Pellegrini et al. firstly described the use of ex vivo cultured limbal epithelial stem cell to treat LSCD. Since then, several reports describing alternative methods of the clinical use of this technology have been published but the retention of stem cells, which is the essential property of the graft, has not been investigated. The definition of a graftable limbal culture in light of the clinical performance must follow specific quality criteria, here discussed, which are relevant for the future use of any cultured cell type for clinical application.
2014
- Customizing Properties of β-Chitin in Squid Pen (Gladius) by Chemical Treatments
[Articolo su rivista]
Ianiro, Alessandro; Di Giosia, Matteo; Fermani, Simona; Samorì, Chiara; Barbalinardo, Marianna; Valle, Francesco; Pellegrini, Graziella; Biscarini, Fabio; Zerbetto, Francesco; Calvaresi, Matteo; Falini, Giuseppe
abstract
The squid pen (gladius) from the Loligo vulgaris was used for preparation of β-chitin materials characterized by different chemical, micro- and nano-structural properties that preserved, almost completely the macrostructural and the mechanical ones. The β-chitin materials obtained by alkaline treatment showed porosity, wettability and swelling that are a function of the duration of the treatment. Microscopic, spectroscopic and synchrotron X-ray diffraction techniques showed that the chemical environment of the N-acetyl groups of the β-chitin chains changes after the thermal alkaline treatment. As a consequence, the crystalline packing of the β-chitin is modified, due to the intercalation of water molecules between β-chitin sheets. Potential applications of these β-chitin materials range from the nanotechnology to the regenerative medicine. The use of gladii, which are waste products of the fishing industry, has also important environmental implications. - See more at: http://www.mdpi.com/1660-3397/12/12/5979/htm#sthash.7aVdC53H.dpuf
2014
- Development of allele-specific gene-silencing siRNAs for TGFBI Arg124Cys in lattice corneal dystrophy type I.
[Articolo su rivista]
Courtney, Dg; Atkinson, Sd; Moore, Je; Maurizi, E; Serafini, Chiara; Pellegrini, Graziella; Black, Gc; Manson, Fd; Yam, Gh; Macewen, Cj; Allen, Eh; Mclean, Wh; Moore, Cb
abstract
PURPOSE:
This study aimed to investigate the potency and specificity of short-interfering RNA (siRNA) treatment for TGFBI-Arg124Cys lattice corneal dystrophy type I (LCDI) using exogenous expression constructs in model systems and endogenous gene targeting in an ex vivo model using corneal epithelial cell cultures.
METHODS:
A panel of 19 TGFBI-Arg124Cys-specific siRNAs were assessed by a dual-luciferase reporter assay. Further assessment using pyrosequencing and qPCR was used to identify the lead siRNA; suppression of mutant TGFBIp expression was confirmed by Western blot and Congo red aggregation assays. An ex vivo model of LCDI was established using limbal biopsies from corneal dystrophy patients harboring the Arg124Cys mutation. Treatment efficiency of the siRNA was assessed for the inhibition of the mutant allele in the primary patient's corneal epithelial cells using pyrosequencing, quantitative PCR (qPCR), and an ELISA.
RESULTS:
A lead siRNA was identified, and demonstrated to be potent and specific in inhibiting the TGFBI-Arg124Cys mutant allele at the mRNA and protein levels. Besides high allele specificity, siRNA treatment achieved a 44% reduction of the endogenous Arg124Cys allele in an ex vivo model of LCDI.
CONCLUSIONS:
We have identified a lead siRNA specific to the TGFBI-Arg124Cys mutant allele associated with LCDI. Silencing of exogenous TGFBI was observed at mRNA and protein levels, and in an ex vivo model of LCDI with an efficient suppression of the endogenous mutant allele. This result indicates the potential of siRNA treatment as a personalized medicine approach for the management of heritable TGFBI-associated corneal dystrophies.
2014
- Eyes on the prize: limbal stem cells and corneal restoration. [5YIF: 24,57; Citations: 5]
[Articolo su rivista]
Pellegrini, Graziella; DE LUCA, Michele
abstract
Corneal diseases and blindness can result from deficiency in limbal stem cells, and autologous transplantation is often the only therapeutic option. Two recent studies in Nature have provided insights into regulation of limbal stem cell function and corneal regeneration and present new opportunities for clinical interventions in eye diseases.
2014
- Identifying the role of matrix metalloproteinases in the pathomechanism of TGFBI Arg124Cys related Lattice Corneal Dystrophy Type I
[Relazione in Atti di Convegno]
Moore, Johnny E.; Courtney, David G.; Atkinson, Sarah D.; Maurizi, Eleonora; Nesbit, Andrew M.; Pellegrini, Graziella; Azar, Dimitri T.; Mclean, Irwin W.; Moore, Tara C. B.
abstract
2014
- Long-term stability and safety of transgenic cultured epidermal stem cells in gene therapy of junctional epidermolysis bullosa.
[Articolo su rivista]
DE ROSA, Laura; Carulli, S; Cocchiarella, Fabienne; Quaglino, Daniela; Enzo, Elena; Franchini, Eleonora; Giannetti, A; DE SANTIS, Giorgio; Recchia, Alessandra; Pellegrini, Graziella; DE LUCA, Michele
abstract
We report a long-term follow-up (6.5 years) of a phase I/II clinical trial envisaging the use of autologous genetically modified cultured epidermal stem cells for gene therapy of junctional epidermolysis bullosa, a devastating genetic skin disease. The critical goals of the trial were to evaluate the safety and long-term persistence of genetically modified epidermis. A normal epidermal-dermal junction was restored and the regenerated transgenic epidermis was found to be fully functional and virtually indistinguishable from a normal control. The epidermis was sustained by a discrete number of long-lasting, self-renewing transgenic epidermal stem cells that maintained the memory of the donor site, whereas the vast majority of transduced transit-amplifying progenitors were lost within the first few months after grafting. These data pave the way for the safe use of epidermal stem cells in combined cell and gene therapy for genetic skin diseases.
2014
- siRNA silencing of the mutant keratin 12 allele in corneal limbal epithelial cells grown from patients with Meesmann's epithelial corneal dystrophy.
[Articolo su rivista]
Courtney, Dg; Atkinson, Sd; Allen, Eh; Moore, Je; Walsh, Cp; Pedrioli, Dm; Macewen, Cj; Pellegrini, Graziella; Maurizi, E; Serafini, Chiara; Fantacci, Monica; Liao, H; Irvine, Ad; Mclean, Wh; Moore, Cb
abstract
PURPOSE:
The aim of this study is to further assess our previously reported keratin 12 (K12)-Leu132Pro specific siRNA in silencing the mutant allele in Meesmann's Epithelial Corneal Dystrophy (MECD) in experimental systems more akin to the in vivo situation through simultaneous expression of both wild-type and mutant alleles.
METHODS:
Using KRT12 exogenous expression constructs transfected into cells, mutant allele specific knockdown was quantified using pyrosequencing and infrared Western blot analysis, while the silencing mechanism was assessed by a modified rapid amplification of cDNA ends (5'RACE) method. Corneal limbal biopsies taken from patients suffering from MECD were used to establish cultures of MECD corneal limbal epithelial stem cells and the ability of the siRNA to silence the endogenous mutant KRT12 allele was assessed by a combination of pyrosequencing, qPCR, ELISA, and quantitative-fluorescent immunohistochemistry (Q-FIHC).
RESULTS:
The siRNA displayed a potent and specific knockdown of K12-Leu132Pro at both the mRNA and protein levels with exogenous expression constructs. Analysis by the 5'RACE method confirmed siRNA-mediated cleavage. In the MECD cells, an allele-specific knockdown of 63% of the endogenous mutant allele was observed without effect on wild-type allele expression.
CONCLUSIONS:
Combined with an effective delivery vehicle this siRNA approach represents a viable treatment option for prevention of the MECD pathology observed in K12-Leu132Pro heterozygous individuals.
2013
- Biological parameters determining the clinical outcome of autologous cultures of limbal stem cells
[Articolo su rivista]
Pellegrini, Graziella; Rama, P; Matuska, S; Lambiase, A; Bonini, S; Pocobelli, A; Colabelli, Rg; Spadea, L; Fasciani, R; Balestrazzi, E; Vinciguerra, P; Rosetta, P; Tortori, A; Nardi, M; Gabbriellini, G; Traverso, Ce; Macaluso, C; Losi, Lorena; Percesepe, Antonio; Venturi, Beatrice; Corradini, Francesca; Panaras, Athanasios; DI ROCCO, Antonio; Guatelli, P; DE LUCA, Michele
abstract
Aim: Limbal cultures restore the corneal epithelium in patients with ocular burns. We investigated the
biological parameters instrumental for their clinical success. Methods: We report a long-term multicenter
prospective study on 152 patients carrying corneal destruction due to severe ocular burns, treated with
autologous limbal cells cultured on fibrin and clinical-grade 3T3-J2 feeder cells. Clinical results were statistically
evaluated both by parametric and nonparametric methods. Results: Clinical outcomes were scored as full
success, partial success and failure in 66.05, 19.14 and 14.81% of eyes, respectively. The total number of
clonogenic cells, colony size, growth rate and presence of conjunctival cells could not predict clinical results.
Instead, the clinical data provided conclusive evidence that graft quality and likelihood of a successful outcome
rely on an accurate evaluation of the number of stem cells detected before transplantation as holoclones
expressing high levels of the p63 transcription factor. No adverse effects related to the feeder layer have
been observed and the regenerated epithelium was completely devoid of any 3T3‑J2 contamination.
Conclusion: Cultures of limbal stem cells can be safely used to successfully treat massive destruction of the
human cornea. We emphasize the importance of a discipline for defining the suitability and the quality of
cultured epithelial grafts, which are relevant to the future clinical use of any cultured cell type.
2013
- Isolation of human keratinocyte stem cells and high-throughput screening approach for their characterization
[Abstract in Atti di Convegno]
Di Rocco, Antonio; Carulli, Sonia; Tenedini, Elena; Bianchi, Elisa; Tagliafico, Enrico; Manfredini, Rossella; Pellegrini, Graziella; DE LUCA, Michele
abstract
In the last three decades, regenerative medicine has opened new horizons for the in vitro
reconstruction of epithelial tissues and gene therapy treatment of skin disorders involving the
use of adult keratinocyte stem cells (KSCs). Although the ability to identify and isolate these cells
represents an important prerequisite for the development of these approaches, molecular markers
and their precise in vivo localization are still lacking. In order to define genes involved in the control
of stemness and commitment of KSCs, we developed a non-invasive, stem cell-preserving magnetic
micro beads based method in order to obtain a KSCs enriched population for high throughput
screening experiments. After 3T3 murine fibroblast feeder layer depletion from our keratinocyte
cultures, we isolated a subpopulation of basal epithelial cells on the basis of the different expression
levels of the a6β4 integrin. By using different approaches, including clonal analysis and p63 bright cells
quantification, we clearly showed that a6β4 integrin bright cells have greater growth potential and
clonogenic capacity compared to the remaining cell fraction and they include the KSCs population.
Comparing gene expression profile of a KSCs-enriched and a terminally differentiated cell population
coming from the same original primary cell culture we defined a set of genes most probably involved
in stemness maintenance. Ongoing gene profiling on single clone type will allow us to validate this
gene signature and to start functional studies on selected genes. Extending this approach to different
ectodermal derived tissues will provide a genome wide signature of the molecular pathways underlying
self-renewal, commitment and differentiation of KSCs.
2013
- The long and winding road that leads to a cure for epidermolysis bullosa
[Articolo su rivista]
Carulli, Sonia; Contin, Roberta; DE ROSA, Laura; Pellegrini, Graziella; DE LUCA, Michele
abstract
Inherited epidermolysis bullosa (EB) is a family of rare genetic skin disorders characterized by structural and mechanical fragility of skin and mucosal membranes. The main feature of EB is the presence of recurrent skin blistering or erosions, which have a profound impact in the quality of life of EB patients and, in the most severe forms, cause early lethality. During the past two decades, it became possible to identify mutations in genes responsible for different types of EB and characterize the abnormalities of the related proteins. Nowadays, there is no cure for EB; all the treatments are palliative and focused on the relief of the devastating EB clinical picture. Recent advancements in molecular biology, stem cell biology and regenerative medicine have fostered new therapeutic approaches for EB. This review is focused on recent developments in gene therapy, protein replacement and cell-based therapy for EB, all aimed at finding a cure for this devastating disease.
2012
- Device for ocular sampling, particularly for peripheral corneal sampling
[Brevetto]
Desogus, Michele; Pellegrini, Graziella
abstract
Dispositivo per l’esecuzione di prelievi oculari, particolarmente adatto per prelievi corneali periferici.
2012
- Methods for characterization/manipulation of human corneal stem cells and their applications in regenerative medicine.
[Capitolo/Saggio]
Corradini, Francesca; Venturi, Beatrice; Pellegrini, Graziella; DE LUCA, Michele
abstract
Cell therapy is an emerging therapeutic strategy aimed at replacing or repairing severely damaged tissues
with cultured cells. Speci fi cally, ocular burns cause depletion of limbal stem cells, which leads to corneal
opaci fi cation 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 D Np63 a .
2011
- Alterations of epithelial stem cell marker patterns in human diabetic corneas and effects of c-met gene therapy
[Articolo su rivista]
M., Saghizadeh; S., Soleymani; A., Harounian; B., Bhakta; S. M., Troyanovsky; W. J., Brunken; Pellegrini, Graziella; A. V., Ljubimov
abstract
PURPOSE:We have previously identified specific epithelial proteins with altered expression in human diabetic central corneas. Decreased hepatocyte growth factor receptor (c-met) and increased proteinases were functionally implicated in the changes of these proteins in diabetes. The present study examined whether limbal stem cell marker patterns were altered in diabetic corneas and whether c-met gene overexpression could normalize these patterns.METHODS:Cryostat sections of 28 ex vivo and 26 organ-cultured autopsy human normal and diabetic corneas were examined by immunohistochemistry using antibodies to putative limbal stem cell markers including ATP-binding cassette sub-family G member 2 (ABCG2), N-cadherin, ΔNp63α, tenascin-C, laminin γ3 chain, keratins (K) K15, K17, K19, β(1) integrin, vimentin, frizzled 7, and fibronectin. Organ-cultured diabetic corneas were studied upon transduction with adenovirus harboring c-met gene.RESULTS:Immunostaining for ABCG2, N-cadherin, ΔNp63α, K15, K17, K19, and β(1) integrin, was significantly decreased in the stem cell-harboring diabetic limbal basal epithelium either by intensity or the number of positive cells. Basement membrane components, laminin γ3 chain, and fibronectin (but not tenascin-C) also showed a significant reduction in the ex vivo diabetic limbus. c-Met gene transduction, which normalizes diabetic marker expression and epithelial wound healing, was accompanied by increased limbal epithelial staining for K17, K19, ΔNp63α, and a diabetic marker α(3)β(1) integrin, compared to vector-transduced corneas.CONCLUSIONS:The data suggest that limbal stem cell compartment is altered in long-term diabetes. Gene therapy, such as with c-met overexpression, could be able to restore normal function to diabetic corneal epithelial stem cells.
2011
- Vision from the right stem. [5YIF: 9.93; Citations: 13]
[Articolo su rivista]
Pellegrini, Graziella; P., Rama; DE LUCA, Michele
abstract
Cultures of limbal cells are a safe and effective treatmentfor the destruction of the human cornea owing to chemicalburns. The essential feature of the graft is the presenceof an adequate number of stem cells, which can bedetermined by the expression of the p63 transcriptionfactor. Here, we will discuss the general principles definingthe rigorous criteria for graftable limbal cultures inlight of their clinical performances. Such criteria mightprove relevant to the future therapeutic use of anycultured cell type.
2010
- Evaluation of molecular markers in corneal regeneration by means of autologous cultures of limbal cells and keratoplasty
[Articolo su rivista]
R. A., Colabelli Gisoldi; A., Pocobelli; C. M., Villani; D., Amato; Pellegrini, Graziella
abstract
PURPOSE:To determine the epithelial phenotype in patients with a limbal stem cell deficiency (LSCD) after ocular surface reconstruction with autologous cultured stem cells. To correlate the epithelial phenotype with the clinical outcome.METHODS:Six eyes affected by LSCD, verified and graded by impression cytology, were treated with an autologous fibrin-cultured limbal stem cell graft. The clinical outcome was defined as a "success" or a "failure," depending on ocular surface stability. To improve their visual function, 4 patients underwent lamellar or penetrating keratoplasty after the stem cell graft. The phenotype of the regenerated corneal epithelium was determined by immunofluorescence of the corneal button to detect CK12, CK3, CK19, and Muc1 as corneal and conjunctival markers.RESULTS:After a mean follow-up of 24 months, 5 cases were defined as successes; 1 case presented an epithelial defect 4 months after grafting and was defined as a failure. Immunofluorescence performed on 4 patients after lamellar and penetrating keratoplasty confirmed the presence of epithelial corneal markers (CK12 and CK3) in 2 of the success cases and the presence of conjunctival markers (CK19 and Muc1) in the 1 failure case. In one of the success cases, both corneal and conjunctival markers were detected on the corneal button. All success cases showed maintenance of marker accounting for high proliferative potential (DeltaNp63alpha) after transplantation.CONCLUSIONS:Autologous cultures of limbal stem cells can regenerate a functional corneal epithelium in patients affected by unilateral LSCD. We showed a correlation between the clinical outcome and the molecular marker expression.
2010
- Human embryonic stem cell-derived keratinocytes: how close to clinics? [5YIF: 24.57; Citations: 8]
[Articolo su rivista]
Pellegrini, Graziella; De Luca, Michele
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.
2010
- Limbal stem-cell therapy and long-term corneal regeneration.
[Articolo su rivista]
Rama, P; Matuska, S; Paganoni, G; Spinelli, A; De Luca, Michele; Pellegrini, Graziella
abstract
BACKGROUND: 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.METHODS: 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.RESULTS: 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.CONCLUSIONS: Cultures of limbal stem cells represent a source of cells for transplantation in the treatment of destruction of the human cornea due to burns.
2010
- Use of magnetically oriented orthogonal collagen scaffolds for hemi-corneal reconstruction and regeneration. [5YIF: 9.3; Citations: 25]
[Articolo su rivista]
N., Builles; H., Janin Manificat; M., Malbouyres; V., Justin; M. R., Rovère; Pellegrini, Graziella; J., Torbet; D. J., Hulmes; C., Burillon; O., Damour; F., Ruggiero
abstract
We recently showed that the highly organized architecture of the corneal stroma could be reproduced using scaffolds consisting of orthogonally aligned multilayers of collagen fibrils prepared using a high magnetic field. Here we show that such scaffolds permit the reconstruction in vitro of human hemi-corneas (stroma + epithelium), using primary human keratocytes and limbal stem cell derived human keratinocytes. On the surface of these hemi-corneas, a well-differentiated epithelium was formed, as determined both histologically and ultrastructurally and by the expression of characteristic markers. Within the stroma, the keratocytes aligned with the directions of the fibrils in the scaffold and synthesized a new extracellular matrix with typical collagen markers and small, uniform diameter fibrils. Finally, in vivo experiments using a rabbit model showed that these orthogonally oriented multi-layer scaffolds could be used to repair the anterior region of the stroma, leading to re-epithelialization and recovery of both transparency and ultrastructural organization.
2009
- Epithelial stem cells in corneal regeneration and epidermal gene therapy. [5YIF: 6.94; Citations: 68]
[Articolo su rivista]
Pellegrini, Graziella; P., Rama; Mavilio, Fulvio; DE LUCA, Michele
abstract
Regenerative medicine refers to innovative therapies aimed at the permanent restoration of diseased tissues and organs. Regeneration of self-renewing tissues requires specific adult stem cells, which need to be genetically modified to correct inherited genetic diseases. Cultures of epithelial stem cells permanently restore severe skin and mucosal defects, and genetically corrected epidermal stem cells regenerate a normal epidermis in patients carrying junctional epidermolysis bullosa. The keratinocyte stem cell is therefore the only cultured stem cell used both in cell therapy and gene therapy clinical protocols. Epithelial stem cell identification, fate and molecular phenotype have been extensively reviewed, but not in relation to tissue regeneration. In this paper we focus on the localization and molecular characterization of human limbal stem cells in relation to corneal regeneration, and the gene therapy of genetic skin diseases by means of genetically modified epidermal stem cells.
2009
- Gene therapy of inherited skin adhesion disorders: a critical overview
[Articolo su rivista]
DE LUCA, Michele; Pellegrini, Graziella; Mavilio, Fulvio
abstract
Gene therapy has the potential to treat devastating inherited diseases for which there is little hope of finding a conventional cure. These include lethal diseases, like immunodeficiencies or several metabolic disorders, or conditions associated with a relatively long life expectancy but poor quality of life and expensive and life-long symptomatic treatments, such as muscular dystrophy, cystic fibrosis and thalassaemia. Skin adhesion defects belong to both groups. For the nonlethal forms, gene therapy, or transplantation of cultured skin derived from genetically corrected epidermal stem cells, represents a very attractive therapeutic option, and potentially a definitive treatment. Recent advances in gene transfer and stem cell culture technology are making this option closer than ever. This paper critically reviews the progress and prospects of gene therapy for epidermolysis bullosa, and the technical and nontechnical factors currently limiting its development.
2009
- In vitro evidence of nerve growth factor effects on human conjunctival epithelial cell differentiation and mucin gene expression. [5YIF: 3.67; Citations: 24]
[Articolo su rivista]
Lambiase, A; Micera, A; Pellegrini, Graziella; Merlo, D; Rama, P; DE LUCA, Michele; Bonini, S; Bonini, S.
abstract
PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
2008
- Corneal regeneration by means of somatic cell therapy
[Capitolo/Saggio]
Pellegrini, Graziella
abstract
The cornea is the anterior surface of the eye and principal optical element; it has multiple specialized functions. The first is to form a protective physical barrier that shields the inner eye from the external environment, defining what belongs to the body from what does not. The second important function lies in its ability to protect itself and many underlying ocular structures from various types of damage, ranging from physical trauma and biochemical
injury to infection by pathogenic organisms to dangerous effects of long-term exposure to light.
Moreover the cornea serves as the main refractive element of the visual system, directing incoming light onto the crystalline lens, which focuses it onto the retina. Refraction depends on the cornea acquiring transparency during embryonic development and maintaining it throughout adult life. Although the cornea appears
to be one clear membrane, it is composed of several discrete layers. The outermost layer is the corneal epithelium that is renewable, transparent and forms a refracting optical surface along with the tear film. Only the corneal tissue is innervated and can signal pain. This epithelium can also signal to underlying layers, strongly affecting their behavior and function.
An important peculiarity of corneal tissue is the total lack of blood vessels, making it a privileged immune site for transplantation.
The past decade has witnessed much major progres in the field of ocular surrace reconstruction. Recent advances in stem cell biology have led to exploration of stem cell-based therapies
to treat a wide range of human diseases. In the ophthalmology field, much hope has been placed on the potential use or these cells to restore sight, particularly in those condition in which which other established treatment has failed and in which visual function has been damaged or lost. Development of modified natural polymers and synthetic polymers either as cell-carriers or for their potential to replace damaged portions of the human cornea account for the most recent updates in the field.
2008
- Correction of Laminin-5 Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors
[Articolo su rivista]
DI NUNZIO, Francesca; Maruggi, Giulietta; Stefano, Ferrari; Enzo Di, Iorio; Poletti, Valentina; Marta, Garcia; Marcela Del, Rio; DE LUCA, Michele; Fernando, Larcher; Pellegrini, Graziella; Mavilio, Fulvio
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.
2008
- Custom phototherapeutic keratectomy and autologous fibrin cultured limbal stem cell autografting: a combined approach.
[Articolo su rivista]
Vinciguerra, P; Albe', E.; Rosetta, P; DI IORIO, E; Pellegrini, Graziella
abstract
no abstract
2008
- Development of a reconstructed cornea from collagen-chondroitin sulfate foams and human cell cultures
[Articolo su rivista]
N. E., Vrana; N., Builles; V., Justin; J., Bednarz; Pellegrini, Graziella; B., Ferrari; O., Damour; D. J., Hulmes; V., Hasirci
abstract
PURPOSE:To develop an artificial cornea, the ability to coculture the different cell types present in the cornea is essential. Here the goal was to develop a full-thickness artificial cornea using an optimized collagen-chondroitin sulfate foam, with a thickness close to that of human cornea, by coculturing human corneal epithelial and stromal cells and transfected human endothelial cells.METHODS:Corneal extracellular matrix was simulated by a porous collagen/glycosaminoglycan-based scaffold seeded with stromal keratocytes and then, successively, epithelial and endothelial cells. Scaffolds were characterized for bulk porosity and pore size distribution. The performance of the three-dimensional construct was studied by histology, immunofluorescence, and immunohistochemistry.RESULTS:The scaffold had 85% porosity and an average pore size of 62.1 microm. Keratocytes populated the scaffold and produced a newly synthesized extracellular matrix as characterized by immunohistochemistry. Even though the keratocytes lost their CD34 phenotype marker, the absence of smooth muscle actin fibers showed that these cells had not differentiated into myofibroblasts. The epithelial cells formed a stratified epithelium and began basement membrane deposition. An endothelial cell monolayer beneath the foam was also apparent.CONCLUSIONS:These results demonstrate that collagen-chondroitin sulfate scaffolds are good substrates for artificial cornea construction with good resilience, long-term culture capability, and handling properties.
2008
- Gene therapy of inherited skin adhesion disorders
[Articolo su rivista]
De Luca, M.; Pellegrini, G.; Mavilio, F.
abstract
Gene therapy is a potential treatment for devastatinginherited diseases for which there is little hope offinding a conventional cure. These include lethal diseaseslike immunodeficiencies and metabolic disorders,and non-lethal conditions associated with poorquality of life and life-long symptomatic treatments,like muscular dystrophy, cystic fibrosis or thalassemia.Skin adhesion defects belong to both groups. For thenon-lethal forms, gene therapy, or transplantation ofcultured skin derived from genetically corrected epidermalstem cells, represents a very attractive therapeuticoption, and potentially a definitive treatment.Recent advances in gene transfer and stem cell culturetechnology are making this option closer than ever.This paper critically reviews the progress and prospectsof gene therapy for skin adhesion defects, andthe technical and non-technical factors currently limitingits development.
2008
- Medicina rigenerativa e nuove frontiere terapeutiche
[Capitolo/Saggio]
Pellegrini, Graziella; Mavilio, Fulvio
abstract
Lo sviluppo delle colture cellulari ha messo in evidenza che le cellule adulte (non più allo stadio embrionale) potevano essere manipolate e dare origine non solo alla proliferazione, che ne aumentava il numero, ma anche al differenziamento che garantiva
che quelle cellule potessero svolgere le funzioni più "specializzate" dei tessuti. Tutto ciò ha aperto prospettive completamente nuove alla medicina e si è fatta strada l'idea di costruire "pezzi di ricambio" o, comunque, imparare a trasmettere informazioni ad un tessuto danneggiato perché questo venisse riparato.
La pionieristica chirurgia del cardiochirurgo C. Barnard ha dimostrato che il cuore di un individuo poteva essere trapiantato in un altro individuo; oggi questo concetto si sta sostituendo con l'idea che si può tentare di ricostruire un tessuto di un paziente in laboratorio, partendo dalle sue stesse cellule. L'uso di cellule "autologhe" (cioè dello stesso individuo e non di un donatore) per ricostruire o modificare le funzioni di un tessuto, evita tutti i problemi di rigetto e la tossicità dei farmaci che, normalmente, si somministrano per ridurre le reazioni di rigetto.
La lista di tessuti che, potenzialmente, possono essere ricostruiti (o ingegnerizzati) è in crescita. Tutto ciò è dovuto in larga parte ai recenti progressi nello studio delle cellule staminali e alla individuazione delle caratteristiche biologiche uniche di queste cellule, sebbene non tutte le terapie basate sulle cellule staminali prevedano la ricostruzione del tessuto in vitro, come ad esempio alcune terapie che utilizzano le cellule staminali neuronali, ma
piuttosto la stimolazione di cellule endogene. Altre terapie, inoltre, prevedono l'uso di cellule staminali geneticamente modificate per produrre sostanze con funzioni terapeutiche.
Bisogna ricordare che, nonostante le aspettative, il passaggio alla applicazione clinica è stato raggiunto, ad oggi, solo in poche aree, in particolare in quelle nelle quali si era sviluppata una conoscenza più approfondita della biologia delle cellule staminali di quel distretto corporeo. I tessuti che oggi sono ricostruiti ed applicati con relativa facilità comprendono una ampia gamma di superfici epiteliali (pelle, cornea, congiuntiva e membrane mucose),
tessuti scheletrici e sistema ematopoietico. Tra questi, quelli che sono stati geneticamente modificati ed utilizzati con successo per applicazione clinica, includono il sistema ematopoietico e gli epiteli. Questi sistemi sono intrinsecamente differenti nella loro capacità
di autorinnovarsi, nella loro fisiologia e nella struttura fisica. La valutazione e lo studio delle diversità intrinseche ad ogni organo o sistema e la conoscenza della regolazione delle sue cellule staminali è essenziale per lo sviluppo di adeguate strategie di intervento clinico nei vari distretti corporei e nelle diverse patologie.
Il capitolo descrive alcuni tra i diversi modelli possibili e alcune problematiche legate all'uso delle due branche principali della medicina rigenerativa: la terapia cellulare e la terapia genica.
2007
- C/EBPδ regulates cell cycle and self-renewal of human limbal stem cells.
[Articolo su rivista]
Barbaro, V; Testa, Anna; DI IORIO, E; Mavilio, Fulvio; Pellegrini, Graziella; DE LUCA, Michele
abstract
Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein (C/EBP), Bmi1, and Np63 identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBP and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBP 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/EBP, but not Np63, 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/EBP 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.
2007
- Towards therapeutic application of ocular stem cells
[Articolo su rivista]
Pellegrini, Graziella; DE LUCA, Michele; Arsenijevic, Y.
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: [1]; Banks-Schlegel S, Kehinde O, Green H. Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet 1981;1:75–8: [2]; 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: [3]]. 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.
2006
- Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.
[Articolo su rivista]
Mavilio, Fulvio; Pellegrini, Graziella; Ferrari, S.; DI NUNZIO, Francesca; Di Iorio, E.; Recchia, Alessandra; Maruggi, Giulietta; Ferrari, G.; Provasi, E.; Bonini, C.; Capurro, S.; Conti, A.; Magnoni, Cristina; Giannetti, Alberto; DE LUCA, Michele
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.
2006
- Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts in response to wound healing
[Articolo su rivista]
V., Barbaro; E., Di Iorio; S., Ferrari; L., Bisceglia; A., Ruzza; DE LUCA, Michele; Pellegrini, Graziella
abstract
PURPOSE. To characterize the expression of the visual system homeobox gene (VSX1) in human corneal keratocytes both in vitro and in vivo. METHODS. The expression of VSX1 was evaluated through semi-quantitative 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. RESULTS. 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. CONCLUSIONS. 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.
2006
- Facciamo il punto sulla medicina rigenerativa della superficie oculare
[Capitolo/Saggio]
Pellegrini, Graziella; DE LUCA, Michele
abstract
Medicina rigenerativa della superficie oculare
2006
- Gene therapy in combination with tissue engineering to treat epidermolysis bullosa
[Articolo su rivista]
S., Ferrari; Pellegrini, Graziella; T., Matsui; Mavilio, Fulvio; DE LUCA, Michele
abstract
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.
2006
- Q-FIHC: Quantification of fluorescence immunohistochemistry to analyse p63 isoforms and cell cycle phases in human limbal stem cells
[Articolo su rivista]
Di Iorio, E; Barbaro, V; Ferrari, S; Ortolani, C; DE LUCA, Michele; Pellegrini, Graziella
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 Delta N alpha 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.
2006
- Regeneration of squamous epithelia from stem cells of cultured grafts
[Articolo su rivista]
DE LUCA, Michele; Pellegrini, Graziella; Green, H.
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.
2006
- Towards a gene therapy clinical trial for epidermolysis bullosa
[Articolo su rivista]
Ferrari, S; Pellegrini, Graziella; Matsui, T; Mavilio, Fulvio; DE LUCA, Michele
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 400000-500000 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.
2005
- Gene therapy approaches for epidermolysis bullosa
[Articolo su rivista]
S., Ferrari; Pellegrini, Graziella; Mavilio, Fulvio; DE LUCA, Michele
abstract
Human epidermis consists of a stratified epithelium mainly composed of keratinocytes and relies on a stem cell compartment to undergo constant regeneration, Genetic mutations affecting the capacity of basal keratinocytes to adhere firmly to the epidermal basement membrane lead to severe. and very often lethal, blistering disorders known as epidermolysis bullosa. Gene therapy represents a promising potential treatment for these devastating inherited disorders. Human epidermal stem cells can be cultivated ex vivo and stably transduced with integrating gene transfer vectors. allowing genetic and, more important, phenotypic correction of the adhesion properties of keratinocytes, Here we will review some of the issues that need to be addressed to make gene therapy a realistic treatment for these disorders, such as (1) which cells should be targeted, (2) which approach (in vivo or ex vivo) should be chosen, and (3) which gene transfer vector (retrovirus, lentivirus, or integrating nonviral strategies,) should be used for stable gene correction. In the last 10 years, many reports have shown that gene transfer approaches to target epidermal stem cells are feasible and able to restore the adhesion properties of primary keratinocytes from patients with epidermolysis bullosa. In addition, tremendous progress has been achieved in culturing epidermal stem cells and generating sheets of stratified epithelium tor permanent coverage of full-thickness bums. Gene modification of stem cells in combination with advanced tissue-engineering techniques could therefore represent a realistic option for patient-4 with epidermolysis bullosa.
2005
- Isoforms of ΔNp63 and the migration of ocular limbal cells in human corneal regeneration
[Articolo su rivista]
Di Iorio, E; Barbaro, V; Ruzza, A; Ponzin, D; Pellegrini, Graziella; DE LUCA, Michele
abstract
The p63 gene generates transactivating and N-terminally truncated transcripts (Delta Np63) 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 a isoform but not beta or gamma. Holoclones derived from the limbus are rich in a, 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 a migrate progressively through the epithelium of the peripheral and central cornea. in the absence of an attached limbus, no a isoform appears in the corneal epithelium. When migrating cells containing the a 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 a 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.
2005
- Separation of keratan-sulfate-derived disaccharides by high-performance liquid chromatography and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection
[Articolo su rivista]
Volpi, Nicola; Maccari, Francesca; S., Ferrari; DE LUCA, Michele; Pellegrini, Graziella
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 wit. h 2-cyanoacetamide and fluorimetric detection of products generated by hydrolysis of this glycosaminoglycan with Bacillus sp. keratanase 11 or Escherichia freundii endo-beta-galactosidase. Following E. freundii endo-p-galactosidase digestion of bovine corneal KS, the monosulfated disaccharide glcNAc6 beta 3(1 -> 3)gal, accounting for approximate to 95% nmol and 50% yield products, is produced. On the contrary, bovine corneal KS treated with endo-beta-N-acetylglucosaminidase (keratanase 11) from Bacillus sp. generates two major products, the monosulfated disaccharide gal beta(1 -> 4)glcNAc6s (approximate to 50% nmol product) and the disulfated disaccharide gal6s beta(I -> 4)glcNAc6s (approximate to 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 approximate to 108 pmol, 50 ng, to 2160 pmol, 1000 ng, for the disaccharide gal beta(I -> 4)glcNAc6s, and from 92 pmol, 50 ng, to 1840 pmol, 1000 ng, for the disaccharide gal6s beta(I -> 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 mu g/ml/ 106 cells and composed of approximate to 71%nmol of disaccharide gal beta(1 -> 4)glcNAc6s and 18%nmol of the disulfated disaccharide gal6s beta(1 -> 4)glcNAc6s having approximate to 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 ( 50 ng) of this glycosaminoglycan and in relation to its biological function and pathological importance.
2004
- Changing the cell source in cell therapy?
[Articolo su rivista]
Pellegrini, Graziella
abstract
No abstract
2004
- Permanent repigmentation of piebaldism by erbium:YAG laser and autologous cultured epidermis
[Articolo su rivista]
Guerra, L; Primavera, G; Raskovic, D; Pellegrini, Graziella; Golisano, O; Bondanza, S; Kuhn, S; Piazza, P; Luci, A; Atzori, F; DE LUCA, Michele
abstract
BACKGROUND: Several surgical techniques have been proposed for the treatment of piebaldism. These procedures, however, are poorly suited for the treatment of large leucodermal lesions, can cause scars and require multiple donor sites. Recently, it has been reported that autologous cultured epidermis induces scarless repigmentation of large vitiligo lesions, using a single small donor site. OBJECTIVES: To induce permanent repigmentation of large achromic lesions in patients suffering from piebaldism by means of autologous cultured epidermal grafts using a rapid, simple and non-invasive surgical procedure. METHODS: Six patients with piebaldism were enrolled in this study. Achromic epidermis was removed by means of appropriately set erbium:YAG laser and autologous cultured epidermal grafts were applied on to the recipient bed. Melanocyte content was evaluated by 3,4-dihydroxyphenylalanine reaction. The percentage of repigmentation was calculated using a semiautomatic image analysis system. RESULTS: Autologous cultured epidermis, bearing a controlled number of melanocytes, induced repigmentation of all piebald lesions. The mean percentage repigmentation was 95.45% (2791.5 cm2 repigmented/2924.2 cm2 transplanted). CONCLUSIONS: Autologous cultured epidermal grafts induce permanent and complete repigmentation of piebald lesions, in the absence of scars. Erbium:YAG laser surgery is a rapid and precise tool for disepithelialization, hence allowing treatment of large piebald lesions during a single surgical operation.
2004
- Telomerase activity is sufficient to bypass replicative senescence in human limbal and conjunctival but not corneal keratinocytes
[Articolo su rivista]
Pellegrini, Graziella; E., Dellambra; P., Paterna; O., Golisano; C. E., Traverso; P., Rama; P., Lacal; DE LUCA, Michele
abstract
The human ocular surface is covered by the conjunctival,corneal and limbal stratified epithelia. While conjunctival stemcells are distributed in bulbar and forniceal conjunctiva,corneal stem cells are segregated in the basal layer of thelimbus, which is the transitional zone between the cornea andthe bulbar conjunctiva. Keratinocyte stem and transientamplifying (TA) cells when isolated in culture give rise toholoclones and paraclones, respectively. Keratinocyte replicativesenescence ensues when all holoclones have generatedparaclones which express high levels of p16INK4a. In the presentstudy, we show that enforced telomerase activity induces thebypass of replicative senescence in limbal and conjunctivalkeratinocytes, without the inactivation of the p16INK4a/Rbpathway or the abrogation of p53 expression. hTERT-transducedlimbal and conjunctival keratinocytes are capable torespond to both growth inhibitory and differentiation stimuli,since they undergo growth arrest in response to phorbol esters,and activate p53 upon DNA damage. Following a sustainedPKC stimulation, occasional clones of p16INK4a-negative cellsemerge and resume ability to proliferate. Telomerase activity,however, is unable to induce the bypass of senescence in cornealTA keratinocytes cultured under the same conditions. Thesedata support the notion that telomere-dependent replicativesenescence is a general property of all human somatic cells,including keratinocytes, and suggest that telomerase activity issufficient to extend the lifespan only of keratinocytes endowedwith high proliferative potentials (which include stem cells), butnot of TA keratinocytes.
2004
- Towards the correction of genetic defect in corneal keratinocytes from patient with macular corneal Dystrophy type II
[Abstract in Rivista]
Ferrari, S; Ferrari, G; Rossi, C; Miccio, Annarita; Volpi, Nicola; Mavilio, Fulvio; Pellegrini, Graziella; De, Luca
abstract
Towards the correction of genetic defect in corneal keratinocytes from patient with macular corneal Dystrophy type II
2003
- Erbium:YAG laser and cultured epidermis in the surgical therapy of stable vitiligo.
[Articolo su rivista]
Guerra, L; Primavera, G; Raskovic, D; Pellegrini, Graziella; Golisano, O; Bondanza, S; Paterna, P; Sonego, G; Gobello, T; Atzori, F; Piazza, P; Luci, A; DE LUCA, Michele
abstract
BJECTIVE: To induce complete and reproducible repigmentation of large "stable" vitiligo lesions by means of autologous cultured epidermal grafts using a rapid, simple, and minimally invasive surgical procedure. DESIGN: Achromic epidermis was removed by means of appropriately settled erbium:YAG laser, and autologous epidermal grafts were applied onto the recipient bed. Melanocyte content was evaluated by dopa reaction. The percentage of repigmentation was calculated using a semiautomatic image analysis system. SETTING: A biosafety level 3-type cell culture facility, a surgical ambulatory department, and a dermatological department in a hospital. PATIENTS: Twenty-one patients with different types of vitiligo were admitted to the study and treated with autologous cultured epidermal grafts. Inclusion criteria were failure of at least 2 standard medical approaches; no therapy for at least 12 months; no progression of old lesions or appearance of new lesions; no Koebner phenomenon within the past 18 months; and no autoimmune disorders. RESULTS: The average percentage of repigmentation in 21 patients was 75.9% (1759.7 cm2 repigmented/2315.8 cm2 transplanted). Three patients showed a reactivation of their vitiligo and did not show repigmentation. The remaining 18 patients, with 43 distinct lesions, showed an average percentage of repigmentation of 90% (1759.7 cm2 repigmented/1953.4 cm2 transplanted). CONCLUSIONS: Under appropriate conditions, cultured epidermal grafts induce complete repigmentation of stable vitiligo lesions. Erbium:YAG laser surgery can supply a fast and precise tool for disepithelialization, hence allowing treatment of large vitiligo lesions during a single surgical operation.
2002
- Terapia cellulare e genica con cheratinociti umani coltivati
[Capitolo/Saggio]
Pellegrini, Graziella; DE LUCA, Michele
abstract
Dopo una breve introduzione sulle cellule staminali epiteliali, il capitolo analizza varie tipologie di terapia cellulare (copertura permanente delle ustioni estese a tutto spessore, copertura permanente di superfici oculari danneggiate, trattamento di vitiligine "stabile" con autografts coltivati epidermici) e la terapia genica ex vivo dell'epidermolisi bullosa giunzionale.
2002
- Toward gene therapy of junctional epidermolysis bullosa (JEB)
[Abstract in Atti di Convegno]
DE LUCA, Michele; Dellambra, E; Pellegrini, Graziella; Guerra, L; Bondanza, S; Mavilio, Fulvio
abstract
Non disponibile
2001
- Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency
[Articolo su rivista]
Rama, P.; Bonini, S.; Lambiase, A.; Golisano, O.; Paterna, P.; DE LUCA, Michele; Pellegrini, Graziella
abstract
BACKGROUND: Ocular burns cause depletion of limbal stem cells, which leads to corneal opacification and visual loss. Autologous cultured epithelial cells can restore damaged corneas, but this technology is still developing. We sought to establish a culture system that allows preservation of limbal stem cells and preparation of manageable epithelial sheets and to investigate whether such cultures can permanently restore total limbal stem cell deficiency. METHODS: We selected a homogeneous group of patients whose limbal cell deficiency was evaluated by scoring the gravity of the clinical picture and the keratin expression pattern. Stem cells, obtained from the limbus of the contralateral eye, were cultivated onto a fibrin substrate and their preservation was evaluated by clonal analysis. Fibrin cultures were grafted onto damaged corneas. RESULTS: Fibrin-cultured limbal stem cells were successful in 14 of 18 patients. Re-epithelialization occurred within the first week. Inflammation and vascularization regressed within the first 3-4 weeks. By the first month, the corneal surface was covered by a transparent, normal-looking epithelium. At 12-27 months follow-up, corneal surfaces were clinically and cytologically stable. Three patients had a penetrating keratoplasty approximately 1 year after restoration of their corneal surface. Their visual acuity improved from light perception or counting fingers to 0.8-1.0. CONCLUSIONS: Preservation of limbal stem cells in culture gives new perspectives on the treatment of ocular disorders characterized by complete limbal stem cell deficiency. The multicenter nature of this study and the handiness and ease of long-distance transportation of the fibrin-cultured epithelial sheets suggest that this technology can now be widely applied.
2001
- In vitro reconstituted sheets of human corneal epithelium and method of producing the same
[Brevetto]
DE LUCA, Michele; Pellegrini, Graziella
abstract
A method of reconstituting sheets of human corneal epithelium in vitro to be used in grafts by culturing limbar stem cells and the method for selection and propagation of said cell lines on a fibrin substrate.
2001
- In vitro reconstitution of trasplantable sheets of ocular surface epithelium and methods for producing the same
[Brevetto]
Pellegrini, Graziella; DE LUCA, Michele
abstract
concessione USA n° 6,610,538
2001
- Keratinocyte-mediated cell and gene therapy
[Capitolo/Saggio]
DE LUCA, Michele; Guerra, L; Dellambra, E; Pellegrini, Graziella
abstract
Although some markers for the epidermal stem cell compartment have been proposed, their role in specifically identifying keratinocyte stem cells is still controversial.
Therefore, the identification of surface epithelial stem cells relies on either the evaluation of their proliferative capacity or on the identification of slow cycling, [3H]TdR- and BrdU-retaining cells, the latter being feasible only on laboratory animals. The proliferative capacity of human lining epithelial stem cells can be evaluated in vitro by means of clonal analysis. Indeed, three types of keratinocytes with different capacities for multiplication have been identified and isolated in human epidermis, hair follicle, limbal-corneal and conjunctival epithelia, i.e. holoclones, meroclones and paraclones. The authors have recently demonstrated
that cultured autografts bearing holoclones can indeed rapidly and permanently cover a large body surface. Preparation of the wound bed and maintenance
of epidermal stem cells in culture are found to be crucial to the clinical success of the technology. The implications and clincial results of permanent coverage of massive full-thickness burns, treatment of "stable" vitiligo with cultured epidermal autografts and permanent coverage of damaged ocular surfaces after complete loss of the corneal-limbal epithelium as well as ex vivo gene therapy of junctional epidermolysis bullosa are reported in this study and literature review. Basic "quality controls" of the culture system may eliminate one important hitherto uncontrolled variable in the evaluation of cultured autograft clinical performance and should represent a starting point for improving epithelial cultivation, in order to achieve satisfactory and reproducible clinical result.
2001
- Reconstructed laminae of human epithelium corneae and method of producing the same
[Brevetto]
DE LUCA, Michele; Pellegrini, Graziella
abstract
A method of reconstructing laminae of human epithelium corneae in vitro to be used in grafts from cultures of limbar stem cells as well as a method of selecting and transferring such cells to fibrin substrate.
2001
- p63 identifies keratinocyte stem cells
[Articolo su rivista]
Pellegrini, Graziella; E., Dellambra; O., Golisano; E., Martinelli; I., Fantozzi; S., Bondanza; D., Ponzin; F., Mckeon; DE LUCA, Michele
abstract
The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3final sigma has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.
2000
- Toward epidermal stem cell-mediated ex vivo gene therapy of junctional epidermolysis bullosa
[Articolo su rivista]
E., Dellambra; Pellegrini, Graziella; L., Guerra; G., Ferrari; G., Zambruno; Mavilio, Fulvio; DE LUCA, Michele
abstract
Junctional epidermolysis bullosa (JEB) is a group of severe, inherited skin diseases caused by mutations in the genes encoding laminin 5 or other components of the hemidesmosome. Since human epidermis is a self-renewing tissue, gene therapy of JEB requires the stable integration of the transgene into the genome of the epidermal stem cell. Human epidermal stem cells can indeed be cultivated and stably transduced with replication-defective retroviral vectors, allowing full phenotypic correction of the adhesion properties of JEB keratinocytes. Epidermal stem cells generate cohesive sheets of stratified epithelium suitable for the permanent coverage of massive skin defects, and genetically modified epidermal sheets maintain long-term expression of the transgene after transplantation on immunodeficient animals. Moreover, we have developed a clinical procedure that allows transplantation of cultured epidermal sheets on large body areas under local anesthesia and without cicatricial outcomes. Thus, (1) the possibility of cultivating lining epithelia, (2) the availability of noninvasive surgical procedures that allow the grafting of large skin areas, and (3) the demonstration of sustained transgene expression in vitro and in vivo by epidermal stem cells, prompt us to propose the implementation of a phase I/II clinical trial aimed at the ex vivo gene therapy of selected JEB patients. The aim of the trial is to validate the ex vivo procedure in a clinical setting, to prove its overall safety, and to analyze critical issues such as long-term survival of the genetically modified implant, immune response against the transgene product, and persistence of transgene expression at therapeutic levels.PMID: 11084687 [PubMed - indexed for MEDLINE]
2000
- Treatment of 'stable' vitiligo by Timedsurgery transplantation of cultured epidermal autografts
[Articolo su rivista]
L., Guerra; S., Capurro; F., Melchi; G., Primavera; S., Bondanza; R., Cancedda; A., Luci; DE LUCA, Michele; Pellegrini, Graziella
abstract
AbstractObjective: To optimize melanocyte/keratinocyte cocultivation and to evaluate the effectiveness of autologous cultured epidermal grafts in the surgical treatment of stable vitiligo. Design: After optimization of melanocyte/keratinocyte cultures, achromic lesions were disepithelialized by means of programmed diathermosurgery (Timedsurgery) and covered with autologous epidermal grafts prepared from secondary cultures. Melanocyte content was evaluated by dopa reaction. The percentage of repigmentation was calculated using a semiautomatic image analysis system. Setting: A biosafety level 3 cell culture facility and a dermatological department in a hospital. Patients: Thirty-two patients carrying different types of vitiligo were admitted to the study and treated with autologous cultured epidermal grafts. Inclusion criteria were (1) failure of at least 2 standard medical approaches; (2) no therapy for at least 12 months; (3) absence of progression of old lesions, absence of appearance of new lesions, and absence of Koebner phenomenon within the past 18 months; and (4) absence of autoimmune disorders. Results: One hundred five achromic lesions (a total of 6078. 2 cm[2]) were treated. The average percentage of repigmentation, evaluated after 12 to 36 months of follow-up, was 77%. Independent of the type of vitiligo, average percentages of repigmentation of extremities and periorificial sites were 8% (31.8 cm[2] repigmented/ 420.5 cm[2] transplanted) and 35% (17.6 cm[2] repigmented/50.0 cm[2] transplanted), respectively. Percentages of repigmentation of all other body sites ranged from 88% to 96% (4329.7 cm[2] repigmented/4675.2 cm[2] transplanted). Color matching was good and scar formation was not observed. Conclusion: Cultured epidermal grafts can be considered a real therapeutic surgical alternative for stable but not lip-tip vitiligo.
1999
- Analysis of the mechanical properties of in vitro reconstructed epidermis: preliminary results
[Articolo su rivista]
Chistolini, P; De Angelis, G; DE LUCA, Michele; Pellegrini, Graziella; Ruspantini, I.
abstract
Human epidermis can be reconstructed in vitro and is currently used in autografts for the treatment of severe, extensive burns and pigmentation disorders. However, there are neither international standards nor a common nomenclature for engineered tissues. The paper discusses the results of a preliminary study on human cultured epidermis to assess its mechanical tensile strength, and to eventually establish mechanical evaluation criteria that will enable test and comparison of the behaviour of different engineered tissue products. To perform uniaxial tension tests a traditional testing machine was adapted, and dedicated sample holding frame and grips designed.
1999
- Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. [5YIF: 10.77; Citations: 429]
[Articolo su rivista]
Pellegrini, Graziella; Golisano, O; Paterna, P; Lambiase, A; Bonini, S; Rama, P; DE LUCA, Michele
abstract
We have analyzed the proliferative and differentiation potential of human ocular keratinocytes. Holoclones, meroclones, and paraclones, previously identified in skin, constitute also the proliferative compartment of the ocular epithelium. Ocular holoclones have the expected properties of stem cells, while transient amplifying cells have variable proliferative potential. Corneal stem cells are segregated in the limbus, while conjunctival stem cells are uniformly distributed in bulbar and forniceal conjunctiva. Conjunctival keratinocytes and goblet cells derive from a common bipotent progenitor. Goblet cells were found in cultures of transient amplifying cells, suggesting that commitment for goblet cell differentiation can occur late in the life of a single conjunctival clone. We found that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at rather precise times of their life history, namely at 45-50 cell doublings and at approximately 15 cell doublings before senescence. Thus, the decision of conjunctival keratinocytes to differentiate into goblet cells appears to be dependent upon an intrinsic "cell doubling clock. " These data open new perspectives in the surgical treatment of severe defects of the anterior ocular surface with autologous cultured conjunctival epithelium.
1999
- The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin. [5YIF: 3.60, Citations: 215]
[Articolo su rivista]
Pellegrini, Graziella; R., Ranno; G., Stracuzzi; S., Bondanza; L., Guerra; G., Zambruno; G., Micali; DE LUCA, Michele
abstract
BACKGROUND: Cell therapy is an emerging therapeutic strategy aimed at replacing or repairing severely damaged tissues with cultured cells. Epidermal regeneration obtained with autologous cultured keratinocytes (cultured autografts) can be life-saving for patients suffering from massive full-thickness burns. However, the widespread use of cultured autografts has been hampered by poor clinical results that have been consistently reported by different burn units, even when cells were applied on properly prepared wound beds. This might arise from the depletion of epidermal stem cells (holoclones) in culture. Depletion of holoclones can occur because of (i) incorrect culture conditions, (ii) environmental damage of the exposed basal layer of cultured grafts, or (iii) use of new substrates or culture technologies not pretested for holoclone preservation. The aim of this study was to show that, if new keratinocyte culture technologies and/or "delivery systems" are proposed, a careful evaluation of epidermal stem cell preservation is essential for the clinical performance of this life-saving technology.
METHODS:
Fibrin was chosen as a potential substrate for keratinocyte cultivation. Stem cells were monitored by clonal analysis using the culture system originally described by Rheinwald and Green as a reference. Massive full-thickness burns were treated with the composite allodermis/cultured autograft technique.
RESULTS:
We show that: (i) the relative percentage of holoclones, meroclones, and paraclones is maintained when keratinocytes are cultivated on fibrin, proving that fibrin does not induce clonal conversion and consequent loss of epidermal stem cells; (ii) the clonogenic ability, growth rate, and long-term proliferative potential are not affected by the new culture system; (iii) when fibrin-cultured autografts bearing stem cells are applied on massive full-thickness burns, the "take" of keratinocytes is high, reproducible, and permanent; and (iv) fibrin allows a significant reduction of the cost of cultured autografts and eliminates problems related to their handling and transportation.
CONCLUSION:
Our data demonstrate that: (i) cultured autografts bearing stem cells can indeed rapidly and permanently cover a large body surface; and (ii) fibrin is a suitable substrate for keratinocyte cultivation and transplantation. These data lend strength to the concept that the success of cell therapy at a clinical level requires cultivation and transplantation of stem cells. We therefore suggest that the proposal of a culture system aimed at the replacement of any severely damaged self-renewing tissue should be preceded by a careful evaluation of its stem cell population.
1998
- Applicazioni cliniche dei cheratinociti umani normali coltivati in vitro
[Capitolo/Saggio]
DE LUCA, Michele; Guerra, L; Pellegrini, Graziella
abstract
Con la tecnologia messa a punto nel 1975 da Rheinwald e Green (Harvard Medical School, Boston) è possibile coltivare in vitro i cheratinociti umani normali ed ottenere lamine di epitelio pluristratificato con le stesse caratteristiche biochimiche, morfologiche e funzionali dell'epitelio in vivo. tale metodica prevede il prelievo dal paziente di un piccolo frammento di cute sana (2-4 cm quadrati) da cui si ottiene una sospensione cellulare epiteliale che viene posta in coltura primaria. le colture primarie vengono, a loro volta, amplificate in colture secondarie: dalle colture secondarie confluenti si ottengono le lamine di epidermide ricostruita in vitro. Dal 1981, anno in cui vennero effettuati con successo i primi trapianti con lembi di epidermide autologa coltivata in vitro in pazienti ustionati, tale tecnologia ha acquisito una rilevanza clinica sempre maggiore e si è e stasa al trattamento di numerose altre patologie. La messa a punto dei protocolli chirurgici per il trapianto dei lembi epidermici ricostruiti in vitro e la possibilità di effettuare una traduzione stabile di chertinociti staminali, hanno aperto nuove prospettive nel campo della terapia genica non solo di alcune patologie dermatologiche ma anche di particolari malattie sistemiche.
1998
- Corrective transduction of human epidermal stem cells in laminin-5-dependent Junctional Epidermolysis Bullosa.
[Articolo su rivista]
E., Dellambra; J., Vailly; Pellegrini, Graziella; S., Bondanza; O., Golisano; G., Zambruno; G., Meneguzzi; DE LUCA, Michele
abstract
Laminin-5 is composed of three distinct polypeptides, alpha3, beta3, and gamma2, which are encoded by three different genes, LAMA3, LAMB3, and LAMC2, respectively. We have isolated epidermal keratinocytes from a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, which led to complete absence of the beta3 polypeptide. In vitro, beta3-null keratinocytes were unable to synthesize laminin-5 and to assemble hemidesmosomes, maintained the impairment of their adhesive properties, and displayed a decrease of their colony-forming ability. A retroviral construct expressing a human beta3 cDNA was used to transduce primary beta3-null keratinocytes. Clonogenic beta3-null keratinocytes were transduced with an efficiency of 100%. Beta3-transduced keratinocytes were able to synthesize and secrete mature heterotrimeric laminin-5. Gene correction fully restored the keratinocyte adhesion machinery, including the capacity of proper hemidesmosomal assembly, and prevented the loss of the colony-forming ability, suggesting a direct link between adhesion to laminin-5 and keratinocyte proliferative capacity. Clonal analysis demonstrated that holoclones expressed the transgene permanently, suggesting stable correction of epidermal stem cells. Because cultured keratinocytes are used routinely to make autologous grafts for patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary human epidermal stem cells defective for a structural protein opens new perspectives in the long-term treatment of genodermatoses.
1998
- Cultivation of human keratinocyte stem cells: current and future clinical applications
[Articolo su rivista]
Pellegrini, Graziella; Bondanza, S; Guerra, L.; DE LUCA, Michele
abstract
Cultured human keratinocytes have a wide spectrum of clinical applications. Clinical results reported by several investigators are, however, contradictory. In this review, the authors discuss the biological and surgical issues which play a key role in the clinical outcome of cultured epidermal autografts used for the treatment of massive full-thickness burns. The importance of cultivation of epidermal stem cells and of their transplantation onto a wound bed prepared with donor dermis is emphasised. The paper also reviews recent data showing that: (i) cultured epidermal autografts bearing melanocytes can be used for the treatment of stable vitiligo; (ii) keratinocytes isolated from other lining epithelia, such as oral, urethral and corneal epithelia, can be cultivated and grafted onto patients suffering from disabling epithelial defects; (iii) keratinocyte stem cells can be stably transduced with retroviral vectors and are therefore attractive targets for the gene therapy of genodermatoses.
1997
- Corneal epithelial stem-cell transplantation
[Articolo su rivista]
DE LUCA, Michele; Pellegrini, Graziella
abstract
Authors' reply to Kazuo Tsubota PMID: 9167491
1997
- Long term restoration of human corneal surface with autologous corneal epithelium generated by serial cultivation of limbal epithelial cells.
[Articolo su rivista]
Pellegrini, Graziella; C., Traverso; At, Franzi; M., Zingirian; R., Cancedda; DE LUCA, Michele
abstract
BACKGROUND: Complete loss of the corneal-limbal epithelium leads to re-epithelialisation by bulbar conjunctival cells. Since conjunctival and corneal-limbal epithelial cells represent two different cell lines, this conjunctival healing of the cornea is followed by stromal scarring, decreased visual acuity, and severe discomfort. Unilateral corneal-limbal epithelial defects can be resolved by the transplantation of limbal grafts taken from the uninjured eye. However, this procedure requires a large limbal graft to be taken from the healthy eye, and is not possible for bilateral lesions. We investigated the possibility of restoring the human corneal surface with autologous corneal epithelial sheets generated by serial cultivation of limbal cells.
METHODS:
Cells were cultivated from a 1 mm2 biopsy sample taken from the limbus of the healthy eye of two patients with severe alkali burns, and thus complete loss of the corneal-limbal surface, of one eye. Normal corneal differentiation was tested with a specific biochemical marker. Autologous cultured corneal sheets were then grafted onto the damaged eyes of the two patients. The patients were followed up at more than 2 years after grafting.
FINDINGS:
We have shown that corneal progenitor cells are localised in the limbus, that cultured limbal cells generate cohesive sheets of authentic corneal epithelium, and that autologous cultured corneal epithelium restored the corneal surface of two patients with complete loss of the corneal-limbus epithelium. Long-term follow-up showed the stability of regenerated corneal epithelium and the striking improvement in patients' comfort and visual acuity.
INTERPRETATION:
The cultivation of corneal epithelium might offer an alternative to patients with unilateral lesions and a therapeutic chance to patients with severe bilateral corneal-limbal epithelial defects. Our findings give a new perspective on the treatment of ocular disorders characterised by stem-cell deficiency.
1997
- The importance of epidermal stem cells in keratinocyte-mediated gene therapy
[Articolo su rivista]
DE LUCA, Michele; Pellegrini, Graziella
abstract
Non disponibile
1996
- Effects of itraconazole on normal keratinocytes cultivated from human nail matrix
[Articolo su rivista]
Guerra, L; Tosti, A; DE DONCKER, P; Cameli, N; Pellegrini, Graziella
abstract
non disponibile
1996
- The Ocular Albinism type 1 gene product is a membrane glycoprotein localized to melanosomes.
[Articolo su rivista]
M. V., Schiaffino; C., Baschirotto; Pellegrini, Graziella; S., Montalti; C., Tacchetti; DE LUCA, Michele; A., Ballabio
abstract
Ocular albinism type 1 (OA1) is an inherited disorder characterized by severe reduction of visual acuity, photophobia, and retinal hypopigmentation. Ultrastructural examination of skin melanocytes and of the retinal pigment epithelium reveals the presence of macromelanosomes, suggesting a defect in melanosome biogenesis. The gene responsible for OA1 is exclusively expressed in pigment cells and encodes a predicted protein of 404 aa displaying several putative transmembrane domains and sharing no similarities with previously identified molecules. Using polyclonal antibodies we have identified the endogenous OA1 protein in retinal pigment epithelial cells, in normal human melanocytes and in various melanoma cell lines. Two forms of the OA1 protein were identified by Western analysis, a 60-kDa glycoprotein and a doublet of 48 and 45 kDa probably corresponding to unglycosylated precursor polypeptides. Upon subcellular fractionation and phase separation with the nonionic detergent Triton X-114, the OA1 protein segregated into the melanosome-rich fraction and behaved as an authentic integral membrane protein. Immunofluorescence and immunogold analyses on normal human melanocytes confirmed the melanosomal membrane localization of the endogenous OA1 protein, consistent with its possible involvement in melanosome biogenesis. The identification of a novel melanosomal membrane protein involved in a human disease will provide insights into the mechanisms that control the cell-specific pathways of subcellular morphogenesis.
1996
- The epidermal keratinocyte
[Capitolo/Saggio]
DE LUCA, Michele; Pellegrini, Graziella; Zambruno, G.
abstract
Description of cell culture methods for epithelial keratinocytes from different body areas.
1994
- Role of integrins in cell adhesion and polarity in normal keratinocytes and human skin pathologies
[Relazione in Atti di Convegno]
DE LUCA, Michele; Pellegrini, Graziella; Zambruno, G; Marchisio, P. C.
abstract
In vitro, normal human keratinocytes reconstitute a differentiated stratified epidermis, maintaining the same gene expression pattern as its in vivo counterpart and are suitable for permanent grafting onto patients. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are also mediated by integrin receptors that are sorted to defined plasma membrane domains. The hemidesmosome-associated integrin alpha 6 beta 4 is sharply localized at the basal surface of basal cells and codistributes with laminin and nicein/kalinin; the alpha 2 beta 1 and alpha 3 beta 1 integrins are enriched laterally and play crucial roles in cell-cell interaction and proper colony morphology. During wound healing, proliferating and migrating keratinocytes express on their plasma membrane alpha v beta 5 and alpha 5 beta 1, which allow keratinocyte attachment and migration over the provisional matrix present in the wound. TGF beta, which is an autocrine and paracrine mediator in wound healing, specifically increases the synthesis and expression of alpha v beta 5 and alpha 5 beta 1, induces the de novo expression of alpha v beta 6, and determines the loss of integrin polarization. In hyperproliferative skin diseases, such as skin cancer or psoriasis vulgaris, and in normal keratinocytes forced into more frequent cell cycles, the polarized expression of integrins is lost, and alpha 5 beta 1 becomes costitutively expressed on the plasma membrane. In addition, the alpha 6 beta 4 integrin becomes associated with focal contacts. Nerve growth factor (NGF) is a potent autocrine stimulator of keratinocyte growth and induces melanocyte migration toward the leading edge of a healing wound
1993
- CRYOPROTECTIVE AQUEOUS SOLUTIONS USEFUL FOR THE PRESERVATION OF IN VlTRO CULTURED EPITHELIAL SHEETS
[Brevetto]
G., Geraci; M., DE ROSA; M., Rossi; R., Cancedda; DE LUCA, Michele; Pellegrini, Graziella
abstract
A method for the preservation of in vitro expanded epithelial sheets which comprises the incubation of said sheets in a cryopreserving solution characterized by lack of volume increase at the liquid-solid phase transition. The cryopreserving solutions comprise: a) polyethylene glycols having molecular weight not higher than 20 KD; b) at least one cross-linking agent selected from polyols, polyamines, mono- or oligosaccharides, polyethyleneglycols having molecular weight lower than 1 KD .
1993
- Cryoprotective aqueous solutions useful for the preservation of in vitro cultured epithelial cells.
[Brevetto]
Geraci, G.; Rosa, M. DE; Mrossi, M. Rossi; Cancedda, R.; DE LUCA, Michele; Pellegrini, Graziella
abstract
Cryoprotective solutions in which the liquid-solid phase transition occurs without remarkable volume increases are described. The compositions of the invention, in addition to the usual components necessary for the growth and mantainance of biological material, comprise: a) polyethylene glycols (PEG) having molecular weight not higher than 20 kD; b) at least one cross-linking agent selected from polyols, mono- or oligosaccharides, polyethylene glycols having molecular weight lower than 1 kD.
1993
- The basement membrane protein BM-600/nicein codistributes with kalinin and the integrin alpha 6 beta 4 in human cultured keratinocytes
[Articolo su rivista]
MARCHISIO P., C; Cremona, O; Savoia, P; Pellegrini, Graziella; ORTONNE J., P; Verrando, P; Burgeson, R; Cancedda, R; DE LUCA, Michele
abstract
The observation is reported that in low-passage human keratinocyte colonies cultured under conditions that allow full epidermal differentiation (i) the basement membrane protein BM-600/nicein, identified by the mAb GB3, is codistributed with laminin and collagen type IV as well as with the bullous pemphigoid antigen in footprints deposited by growing and migrating colonies; (ii) the integrin heterodimer alpha 6 beta 4 is codistributed with the same molecules suggesting its spatial association with basement membrane components; (iii) the distribution pattern of alpha 6 beta 4 and BM-600/nicein underneath individual cells is identical and is characterized by a typical "leopard skin" pattern complementary to the distribution of submembraneous F-actin microfilament network; (iv) a rabbit polyclonal antiserum to kalinin (R4012) used in double-label immunofluorescence staining with mAb GB3 shows that this protein has the same distribution as BM-600/nicein and this suggests that they are identically located; and (v) immunoprecipitation with mAb GB3 to BM-600/nicein and BM165 to kalinin shows identical bands suggesting that nicein and kalinin represent the same molecular entity. We suggest that alpha 6 beta 4 displays not only an adhesive role for keratinocytes in view of its reported association to hemidesmosomes but may also be involved in organizing the molecules of the epithelial extracellular matrix, including those forming the basement membrane zone and hemidesmosomes, a function proposed for other integrins in other cellular systems.
1992
- Expression, topography and function of integrin receptors is severely altered in keratinocytes from involved and uninvolved psoriatic skin.
[Articolo su rivista]
Pellegrini, Graziella; DE LUCA, Michele; Orecchia, G.; Balzac, F.; Cremona, O.; Savoia, P.; Cancedda, R.; Marchisio, P. C.
abstract
Psoriasis is a hyperproliferative cutaneous disease of unknown etiology and etiopathogenesis. Alteration of keratinocyte adhesiveness to basal lamina has been proposed as the initial disturbance leading to poorly controlled proliferation. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are segregated to discrete membrane domains. In this paper, the expression and function of integrins in psoriatic keratinocytes were examined, both in vivo and in vitro. We found that: (a) in psoriatic keratinocytes the integrin heterodimers alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 have lost their polarized distribution on the plasma membrane; (b) the role of these integrins in mediating keratinocyte adhesion in vitro is altered; (c) psoriatic keratinocytes form focal contacts containing both beta 1 and beta 4 integrins. In normal adult keratinocytes the alpha 5 beta 1 fibronectin receptor is poorly expressed and diffusely distributed on the basal keratinocyte plasma membrane and is not organized in defined adhesive structures. In contrast, psoriatic keratinocytes show a clear fibronectin receptor staining in vivo, and organize alpha 5 beta 1 in typical focal contacts in vitro without any obvious increase of its expression and synthesis. These multiple alterations of integrins are also present in uninvolved keratinocytes from psoriatic patients, suggesting a key role for altered integrin-mediated adhesion in the pathogenesis of this disease.
1992
- In vitro reconstitution of differentiated human epithelia
[Articolo su rivista]
Pellegrini, Graziella; Gherzi, R; Franzi, At; D'Anna, F; DE LUCA, Michele; Cancedda, R.
abstract
N/A
1992
- The control of polarized integrin topography and the organization of adhesion-related cytoskeleton in normal human keratinocytes depend upon number of passages in culture and ionic environment.
[Articolo su rivista]
DE LUCA, Michele; Pellegrini, Graziella; Bondanza, S; Cremona, O; Savoia, P; Cancedda, R; Marchisio, P. C.
abstract
Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are sorted to defined membrane domains. The hemidesmosome-associated integrin alpha 6 beta 4 is sharply localized to the basal surface of basal cells while alpha 2 beta 1 and alpha 3 beta 1 are enriched laterally. This integrin sorting pattern is perfectly reproducible in vitro by cultured keratinocytes and takes place progressively in primary or secondary culture in the presence of 1.8 mM Ca2+. The polarized topography of integrins is gradually lost with higher passage numbers and between passage 5 and passage 7 there is a complete pericellular redistribution of the above integrins. Along with the decreased basal adhesive value of alpha 6 beta 4 there is a marked increase in the number of focal contacts in high-passage keratinocyte colonies. A similar loss of polarized topography of integrins occurs under low-Ca2+ culture conditions. Increasing the number of culture passages beyond the fifth induces the appearance of the fibronectin receptor alpha 5 beta 1 on the surface of keratinocytes, particularly at intercellular junctions and in some focal contacts. The receptor alpha 5 beta 1 is not detectably exposed by low-passage cells. We propose that forcing keratinocytes into more frequent cell cycles by continuous passaging may perturb the polarized topography of integrins and the adhesion mechanisms of keratinocytes. Then, low-passage keratinocytes are, in our opinion, the most reliable in vitro models for studying the physiology of epidermal cells.
1989
- A novel type of GABA receptor in rat spinal cord?
[Articolo su rivista]
Raiteri, M; Pellegrini, Graziella; Cantoni, C. AND BONANNO G.
abstract
The depolarization-evoked release of gamma-aminobutyric acid (GABA) and its possible modulation mediated by autoreceptors were studied in nerve endings isolated from rat spinal cord and prelabeled with the radioactive aminoacid. In the presence of the GABA uptake inhibitor SK&F 89976A [N-(4,4-diphenyl-3-butenyl)-nipecotic acid], used to minimize carrier-mediated homoexchange, exogenous GABA (1-10 mumol/l) decreased in a concentration-dependent way the release of 3H-GABA evoked by 15 mmol/l KCl. The GABAA receptor agonist muscimol (10-100 mumol/l) did not affect the K+ (15 mmol/l)-evoked 3H-GABA release. Similarly ineffective was the GABAB receptor agonist (-)-baclofen (3-100 mumol/l). The effect of GABA was not counteracted by the GABAA receptor antagonists bicuculline,picrotoxin or SR95531 [2-(3'-carbethoxy-2'-propenyl)-3-amino-6-paramethoxy-phenyl-pyr idazinium bromide].(ABSTRACT TRUNCATED AT 250 WORDS)
1989
- GABA B autoreceptors in rat cortex synaptosomes: response under different depolarizing and ionic conditions
[Articolo su rivista]
Bonanno, G; Pellegrini, Graziella; Asaro, D; Fontana, G. AND RAITERI M.
abstract
Rat cerebral cortex synaptosomes prelabeled with [3H]gamma-aminobutyric acid [( 3H]GABA) were exposed in superfusion to various concentrations of KCl (9-50 mM). The evoked release of [3H]GABA reached a plateau at about 35 mM KCl. The K+-induced release was Ca2+-dependent, particularly at the lowest K+ concentrations. The GABAB agonist (-)-baclofen concentration dependently inhibited the release of [3H]GABA evoked by K+; this effect decreased with increasing K+ concentration and disappeared at 35 mM KCl. The GABAA agonist muscimol (1-100 microM) was totally ineffective to inhibit the release of [3H]GABA. Veratrine (1-30 microM) induced the release of [3H]GABA and the effect was tetrodotoxin-sensitive. (-)-Baclofen, but not muscimol, decreased the veratrine-induced [3H]GABA release; the GABAB agonist was particularly effective in presence of low concentrations of veratrine (1-3 microM) but the effect disappeared when 30 microM of the alkaloid was used. The inhibitory effect of (-)-baclofen on the release of [3H]GABA evoked by 15 mM KCl was dependent on the concentration of Ca2+: the effect increased as the concentration of Ca2+ was raised, reaching a plateau at 0.6 mM Ca2+. Exogenous GABA, in presence of the GABA uptake blocker SK & F 89976A, inhibited the release of [3H]GABA evoked by K+; this effect was antagonized by phaclofen. The data support the idea that terminal GABA autoreceptors in the rat cerebral cortex are of the GABAB type.
1989
- Release-regulating autoreceptors of the GABAB-type in human cerebral cortex
[Articolo su rivista]
Bonanno, G; Cavazzani, P; ANDRIOLI G., C; Asaro, D; Pellegrini, Graziella; Raiteri, M.
abstract
1. The depolarization-evoked release of gamma-aminobutyric acid (GABA) and its modulation mediated by autoreceptors were investigated in superfused synaptosomes prepared from fresh human cerebral cortex. 2. The release of [3H]-GABA provoked by 15 mM K+ from human cortex nerve endings was almost totally (85%) calcium-dependent. 3. In the presence of the GABA uptake inhibitor SK&F 89976A (N-(4,4-diphenyl-3-butenyl)-nipecotic acid), added to prevent carrier-mediated homoexchange, GABA (1-10 microM) decreased in a concentration-dependent manner the K+-evoked release of [3H]-GABA. The effect of GABA was mimicked by the GABAB receptor agonist (-)-baclofen (1-100 microM) but not by the GABAA receptor agonist muscimol (1-100 microM). Moreover, the GABA-induced inhibition of [3H]-GABA release was not affected by two GABAA receptor antagonists, bicuculline or SR 95531 (2-(3'-carbethoxy-2'-propenyl)-3-amino-6-paramethoxy-phenyl-pyr idazinium bromide). 4. (-)-Baclofen also inhibited the depolarization-evoked release of endogenous GABA from human cortical synaptosomes. 5. It is concluded that GABA autoreceptors regulating the release of both newly taken up and endogenous GABA are present in human brain and appear to belong to the GABAB subtype.
1988
- Human brain cortex nerve endings are endowed with GABA autoreceptors of the GABAB subtype
[Articolo su rivista]
Bonanno, G; Pellegrini, Graziella; Asaro, D; Ruelle, A; Andrioli, Gc; Raiteri, M.
abstract
No abstract available
1988
- In vitro release of somatostatin from rat cerebral cortex synaptosomes and slices
[Articolo su rivista]
Bonanno, G; Pellegrini, Graziella; Asaro, D; Raiteri, M.
abstract
No abstract available
1987
- Studies on 3H-GABA and endogenous GABA release in rat cerebral cortex suggest the presence of autoreceptors of the GABA B type
[Articolo su rivista]
Pittaluga, A; Asaro, D; Pellegrini, Graziella; Raiteri, M.
abstract
The presence of autoreceptors for gamma-aminobutyric acid (GABA) in the CNS was reinvestigated using rat cortex synaptosomes prelabeled with [3H]GABA and exposed to GABA by superfusion in the presence of a new GABA uptake inhibitor, N-(4,4-diphenyl-3-butenyl)-nipecotic acid (SK&F 89976A). This compound itself did not increase the basal or the depolarization-evoked release of [3H]GABA. GABA reduced in a concentration-dependent way the release of [3H]GABA evoked by 15 mM K+. The effect was not antagonized by bicuculline, picrotoxin or by the new GABAA antagonist SR 95531. The GABAA agonist muscimol did not affect [3H]GABA release. This was reduced by (-)baclofen (but not by the (+) isomer) and the concentration-inhibition curve of (-)baclofen was superimposable on to that of GABA. Also the K+-evoked release of endogenous GABA was stereoselectively and concentration dependently inhibited by the (-) enantiomer of baclofen. It is concluded that the release of GABA from rat cortical nerve endings may be inhibited through the activation of autoreceptors which appear to belong to the GABAB type.