The new therapeutic potential of stem cells in ophthalmology.
Recent advances in regenerative medicine, in particular therapies based on the use of autologous adult stem cell cultures, and the first recently proposed clinical trials with pluripotent stem cells, have generated enthusiasm and much effort to explore the new therapeutic potential of both adult and pluripotent stem cells.
In recent decades, developmental biology has elucidated many cellular and molecular mechanisms that regulate stem cell-dependent tissue homeostasis, making us aware of the molecular basis of many diseases. Many human tissues and organs possess the ability to self-renew and repair acute and chronic injuries. These processes are based on the presence of specific stem cells, which generate progenitors (often referred to as transiently amplifying cells), which generate terminally differentiated cells. Typical examples of these transitions are haematopoietic stem cells, which give rise to all blood cells through myeloid and lymphoid progenitors, or epidermal stem cells, which give rise to the epidermis, hair follicle and sebaceous glands; the mesenchymal stem cells, which derive from the bone marrow, are capable of generating all the tissues found in the bone segment (bone, cartilage, adipocytes, fibroblasts and the stroma that supports haematopoiesis), better defined as skeletal stem cells.
of the corneal epithelium, have been shown to completely restore a severely damaged corneal epithelium and allow recovery of vision in patients with corneal destruction caused by chemical burns. The stem cells are isolated by enzymatic treatment from a 1-2 mm biopsy of limbus, the thin zone between the cornea and conjunctiva. The limbus is the only corneal zone with papillae-like invaginations, called Vogt Palisades e limbal epithelial cryptscontaining many small basal cells that lack keratin 3, which is specific for corneal differentiation. This limbal basal layer contains cells that slowly complete the cell cycle and form holoclones, whereas central corneal cells do not. Many molecular markers have been proposed to identify corneal stem cells, however, correlation with the ability to regenerate the cornea in the long term has only been demonstrated for a few of them.
Shortly after the first report of successful clinical application with cultured limbal stem cells, several dozen similar protocols were proposed with related clinical applications. Investigations into alternative methods and pathologies eligible for treatment have contributed to increasing our knowledge in this field, while questions related to the identification of the causes of variability related to the reagents used for tissue reconstruction, the selection of patients included in the treatment, the drugs used in parallel, surgical and post-operative management, and the implications of these on the success rate, safety and reproducibility of clinical outcomes have increased.
The cultural separation between different scientific fields makes it difficult to establish multidisciplinary criteria that are, however, necessary for optimal translational research. In this article, an attempt is made to propose some criteria (specifically related to both clinical and biological parameters) for translational medicine with cultivated limbal stem cells.
We also offer an extensive bibliography to serve as a guide for those who wish to learn more about this field of research.
Patient selection
The limbal stem cell pool of the corneal epithelium decreases due to hereditary or acquired damage, leading to partial or total limbal stem cell deficiency (LSCD). This definition refers to a heterogeneous group of pathologies that have a deleterious effect on corneal integrity and wound closure. As a consequence of stem cell depletion, invasion of the peripheral and central cornea by conjunctival epithelium occurs. This tissue delocalisation induces neovascularisation of a normally avascular ocular area and induces corneal opacification, resulting in a severe drop in visual acuity or blindness. Conjunctival invasion can be prevented or prevented by the relocalisation of corneal stem cells obtained by transplantation of flaps generated from the culture of autologous limbal cells taken from the undamaged or less damaged eye.
[caption id="attachment_1536" align="alignright" width="178"]
Ph. 2. Bilateral LSD before and after treatment (courtesy of Dr. Paolo Rama).[/caption].
Appropriate selection and preparation of the 'recipient bed' are of great importance for a successful clinical outcome of limbal cultures. Failures may occur due to the severity of the damage, the degree of inflammation and post-operative complications. Chemical burns can damage the eyelids, conjunctiva and lacrimal apparatus. In the course of such damage, the ocular surface is chronically inflamed and the resulting alteration of the microenvironment can impede the take-over of cultured stem cells. In the case of extensive damage of the entire ocular surface, reconstruction of the conjunctiva, which allows movement of the eyeball and physiological distribution of the tear film, must be achieved prior to cell transplantation. In the absence of systemic or genetic negative stimuli, transplantation of autologous limbal cell cultures can itself partially or fully restore the macro- or microenvironment. This may explain why, in ocular surfaces severely damaged by chemical burns, the second transplantation of cultured epithelium produces a higher degree of success than the first transplantation. The first transplantation of cultured cells probably 'normalises the environment' through the production of matrix molecules such as laminin 5, proteoglycans and collagen, the paracrine and autocrine secretion of a balanced amount of growth factors, such as TGF alpha, interleukins, PDGF, Insulin like Growth Factor 1, TGF beta, Nerve Growth Factor, basic Fibroblasts Growth Factor, and epithelial-mesenchymal 'cross-talk'. Despite many years of proven clinical success, we still have no idea how many stem cells can take root on the recipient bed of the lesion. Moreover, ocular surface pathologies attributed to limbal stem cell deficiency may have a variety of aetiologies, but not all of them have been shown to be associated with total limbal stem cell depletion. In fact, they could be characterised by a macro- and micro-environment that is not permissive for appropriate proliferation and differentiation of existing stem cells. Varieties of limbal deficits could provide 'behavioural instructions' to transplanted cells, which guide different cellular responses during or after rooting.
Biological Parameters
Several methods have been proposed for the culture of limbal stem cells. The composition of the culture medium plays an important role in preserving limbal stem cells, and different mixtures of materials and reagents for culture have been proposed. Among the various proposals, it is unclear when Good Manufacturing Practice or GMP (which has come into force in Europe for these products) has been applied to biological reagents intended for the manufacture of advanced therapies (cultured tissues).
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Ph. 3. Colonies of epithelial cells obtained from a stem cell (courtesy of the 'Stefano Ferrari' Centre for Regenerative Medicine).[/caption].
In fact, even the details of the criteria used for the selection of appropriate materials and reagents have not been regularly described and it has never been explained how these materials were considered suitable for maintaining stem cells in culture. In the absence of these data, it becomes difficult to assess the clinical success rate or safety of each developed culture system, and whether the differences are related to the culture technique or the selection of individual reagent batches. Obviously, the comparison of different culture conditions cannot show differences if the stem cells were not maintained in any of the conditions.
In past years, various studies have proposed different combinations of growth factors and hormones in the culture medium. These media can induce stimulation of different metabolic pathways with a variety of cellular responses both in culture and after transplantation. To produce clinical success, a corneal cell culture must contain sufficient numbers of keratinocyte stem cells, which are essential for long-term corneal renewal, rather than a fully stratified epithelium in culture. Clinical results obtained with different techniques may help identify key information on the appropriateness of the culture process.
The problem of xenogenic components
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Ph. 5. A limbal cell in mitosis (courtesy of the 'Stefano Ferrari' Centre for Regenerative Medicine)[/caption].
Culture media often contain animal-derived materials, such as bovine serum. Foetal serum is used in this, as well as in other cell culture models. Some authors have proposed the use of autologous human serum, with the intention of preventing putative xenogenic contaminants, to replace foetal bovine serum. It is, however, necessary to consider the variable content of hormones and growth factors, due to the different genetic characteristics of each individual, which would make the cultures non-reproducible, as they would grow in culture media with a different composition for each patient. These differences may have an impact on the maintenance of stem cells, and would reduce the reliability of culture controls by making it impossible to define specific, well-defined criteria for assessing the quality of cell culture. Frequently used foetal bovine serum is derived from mixtures of different sera, so individual variability in content is minimised; certainly the foetal serum used must be analysed extensively for pathogen absence in accordance with current health regulations. Equivalent use of mixtures of human sera could obviate individual variability, however, the risk of contamination by viruses, infectious or viral agents and prions also exists for reagents of human origin and, especially in the absence of species barriers, anything can be transmitted.
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Ph. 5. Corneal cells seen under a fluorescence microscope (courtesy of the Centre for Regenerative Medicine 'Stefano Ferrari').[/caption]
Another proposed choice is synthetic culture media, which, however, lack a huge number of physiological enhancers, some of which are known and could in future be produced by synthesis and addition. It must be considered, however, that many of these are still unknown and the purification (under GMP conditions) of all the necessary hormones and growth factors would make the cost of the media unattainable. Similar considerations apply to many other elements of culture media, often of animal origin and not yet replaceable if cellular functions are to be maintained, which must be carefully checked before use.
Biomechanical signals and surface modification
A typical strategy, used in stem cell-based therapies, is to reconstruct engineered tissues with cells grown on biomaterials that can 'mimic' the biochemical and biophysical microenvironment in vivo. For this purpose, many different materials have been proposed to cultivate the cells, such as fibrin, amniotic membrane, plastic or polymers of various kinds. The interaction between stem cells, their surrounding microenvironment and external forces (which represent the stem cells' 'niche') must be well understood before therapies can be applied clinically. Cell behaviour is not only governed by chemical signals. Tissue architecture and mechanical forces, to which they are subjected, are dominant signals that govern cellular decisions: cell-matrix and cell-cell adhesion interactions, the organisation of the cytoskeleton, and the tension forces that keep individual cells and the whole tissue in a precise shape, all represent, biological 'architecture' signals. The mechanical connections between the matrix and cytoskeleton allow cells to exert tensile forces that are transmitted to the cell nucleus, the result of which is the triggering of signals that modulate adhesion, surface diffusion, migration, proliferation and differentiation. More generally, mechano-transduction represents a combination of signals, including the juxtaposition of molecular, topographical and mechanical stimuli, present at the interaction interface. To study these interactions, surfaces exposing selected bio-functional molecular groups or specifically engineered micrometre-scale moulds were used. These tests showed that substrate hardness, surface nano-topography, micro geometry and extracellular forces, and even fluid fluxes, can all have an important influence on the regulation of the activities of different cell types. For example, in vivothe formation of skin scars activates Transforming Growth Factors and SMAD signalling, adhesion molecules such as integrins and calcium signals; or in the case of myoblasts, when laid on large strips of soft hydrogel, they decrease their proliferation and increase their fusion index.
Medication during surgery and follow-up
The management of post-traumatic or post-operative problems involves extensive use of drugs. In the case of treatment for limbal stem deficits, the impact of acute and chronic corneal toxicity of eye drop components is not adequately considered in regenerative medicine protocols. In spite of this, a large number of experimental and clinical studies have shown that prolonged use of topical drugs can induce ocular surface changes, causing ocular discomfort, tear film instability, loss of calico cells, inflammation, squamous conjunctival metaplasia, epithelial apoptosis, and subconjunctival fibrosis. Prolonged use of local anaesthetics is associated with delayed corneal re-epithelialisation after injury, altered lubrication and tear film, corneal swelling and disruption of epithelial motility. Lidocaine, one of the most commonly used anaesthetics, already at a dose of 250 mg/ml (below the clinical dose), reduces the effectiveness of normal wound healing. Vasoconstrictors, which are generally used to increase the duration of local anaesthesia, may produce a cytotoxic effect and pigment deposition, as reported in the literature for epinephrine and some commonly used anti-glaucoma drugs.
Depending on dosage and mode of administration, corticosteroids, which are widely used, both systemically and topically, in the treatment of ocular inflammatory conditions, have side effects that include delaying or preventing the closure of corneal lesions. Non-steroidal anti-inflammatory drugs inhibit prostaglandin synthesis and exert an equivalent anti-inflammatory effect. These drugs are increasingly used for their concomitant analgesic effect and efficacy in ocular allergic diseases.
Comparative analysis of corneal toxicity revealed cell damage, altered cell viability, proliferation and migration even after short-term exposure of corneal cells to certain non-steroidal anti-inflammatory drugs. Other commonly used drugs, such as prostaglandin analogues, anti-allergy drops or multipurpose solutions (especially in the presence of preservatives) showed similar toxic effects. In particular, the scientific data suggest an interpretation of irreversible damage of the epithelial proliferative compartment, which includes stem cells; effects on cell viability (with a decrease up to 40%), proliferation, maintenance of corneal thickness, endothelial permeability, DNA integrity and lubrication have been described, due to common preservatives such as benzalkonium chloride (BAC), especially in the case of repeated administration or persistence of these drugs on damaged ocular surfaces.
It is clear that all these toxic effects are amplified when drugs are used prior to the full rooting of a cultured cell population.
In conclusion, no drug appears to be free of toxicity of some kind, so the selection of drugs on applied cell cultures and the harmonisation of dosages and administration protocols must be carefully considered to increase the benefit/risk ratio.
Conclusions
Rapid advances in stem cell research have raised the interest of governments, the media and, of course, patients. Their clinical success depends on factors unique to cell therapies, which include manufacturing procedures, standardisation of clinical and pharmacological protocols, and safety regulation. As already discussed, successful clinical application of any cell therapy protocol requires optimisation of the culture method (especially when stem cells are required) and surgical procedures, control of the microenvironment in which the cells are to take root, and appropriate pharmacological support.
This scenario is further complicated by new regulations on clinical applications of cells and tissues. As with many traditional drugs, cell cultures for clinical application must be obtained in accordance with current Good Manufacturing Practice (GMP) standards, but cell cultures are inherently more complex and less well controlled than a small molecule. Due to their biological nature, living cell products cannot be fully defined with the same methods as traditional drugs. The choice to apply the rules of medicinal products to cell cultures, without specific adaptations for this field, is not entirely acceptable. Indeed, this choice creates problems for scientists and regulators alike. Researchers from both academia and industry can hardly cope with the new European rules on ATMPs (Advanced Therapy Medicinal Products) (EC regulation n°1394). Similar, but not identical, regulation has been applied in the United States and other industrialised countries.
Products based on living cells present many additional challenges especially in today's highly regulated healthcare environment, especially considering that the web of regulations was designed for chemical-type manufacturing in the last century. In addition, there is not much harmonisation between different regulatory authorities, which greatly increases the problems in manufacturing and clinical trials. The international conference for the harmonisation of technical requirements for the registration of pharmaceuticals for human use has only produced agreements on specific topics, such as viral safety and a few others.
Regulation is intended to increase safety, and thus protect patients, and is unquestionable; however, for products based on living cells, it is often achieved at the expense of efficacy, due to a lack of sector-specific adjustments. Every therapy is based on a benefit/risk ratio, so reducing the efficacy of a cell culture below a certain threshold value will generate a safer but useless biological product. Therapies based on the use of living cells, are more complex and inherently less controlled than the synthesis of molecules, due to their biological nature, making this product not fully but only partially defined, according to 'classical' regulations, which require expansion for these reasons. Finally, many patient-specific autologous cell products are 'freshly administered', so release assays cannot all be completed prior to administration, leading us to the idea that the real product is the manufacturing process, and the reliability of manufacture should be periodically assessed through comparison with specific reference cells, determining the limits for the controls that are obtained during tissue manufacture. These limits will be wider for tissue manufacture from different individuals, due to differences in age, sex, lifestyle, and individual genetic characteristics, but should be tighter for large-scale donor treatments.
An understanding of the prospects for regenerative medicine will provide insight into the likely future shape of new therapies, their development timeframes, as well as the planning of the infrastructure required to facilitate its expeditious deployment by academies and the new pharmaceutical industry worldwide, with maximum guarantees for patients.
Graziella Pellegrini
E-mail: graziella.pellegrini@unimore.it
Prof. Graziella Pellegrini is Professor of Biology at the University of Modena and Reggio Emilia and Cell Therapy Coordinator at the 'Stefano Ferrari' Centre for Regenerative Medicine, Prof. Pellegrini has devoted much of her scientific activity to translational medicine and the development of clinical applications of cultured stem cells.
Together with Prof. Michele De Luca, he developed the first treatment with human limbal stem cells for patients with severe corneal burns.
He is one of the founding members of the IOSS (International Ocular Surface Society).Â
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Dr. Carmelo Chines
Direttore responsabile
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