| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
MEETING REPORT |
a Laboratorio Biochimica Clinica, Istituto Superiore Sanità, Rome, Italy;
b Istituto Nazionale per la Ricerca sul Cancro, Centro Biotecnologie Avanzate, Genova, Italy;
c Istituto per la Ricerca contro il Cancro, Torino, Italy
Key Words. Cell therapy • Tissue engineering • Transplantation biology • Stem cells
Anna Rita Migliaccio, Ph.D., Laboratorio Biochimica Clinica, Istituto Superiore Sanità, Viale Regina Elena 299, 00161, Rome, Italy. Telephone: 0039-06-49902576; Fax: 0039-06-49387143; e-mail: migliar{at}iss.it
 |
ABSTRACT |
|---|
 Top Abstract IntroductionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionReferences |
|---|
In October 2001, The Istituto Superiore di Sanità, in collaboration with Istituto Dermatopatico dell’Immacolata, Istituto Nazionale Ricerca Cancro-Centro Biotecnologie Avanzate, and University G. D’Annunzio, organized an international meeting in Rome on cell therapy to provide an overview of the most recent developments in this field and its clinical perspectives. One of the aims of the meeting was to use scientific reports as a backbone against which to review both regulatory issues and progress reports from the first clinical protocols that have been approved in Italy. The meeting included six scientific sessions: Immunotherapy, coordinated by Filippo Belardelli and Maurizio Cianfriglia; Epithelium, coordinated by Michele De Luca and Yann Barrandon; Osteoregeneration, coordinated by Rodolfo Quarto and Andrea Facchini; Hematopoiesis, coordinated by Sergio Amadori and Giovanni Migliaccio; and Future Perspectives, coordinated by Cesare Peschle and Maria Orlando, and was concluded by a round table discussion on regulatory affairs.
Extensive reviews on the sessions on Immunotherapy, Hemopoiesis, and Osteocartilage regeneration will be provided by Belardelli et al., McNiece and W. Fibbe, and Cancedda et al. in respective articles that will be published in a forthcoming issue of STEM CELLS. Italian investigators have reached an excellent scientific level in basic cell biology. This fact was reflected by the excellent presentations held by three young (<30 years of age) investigators: Stefano Santini, from the Istituto Superiore di Sanità, Sandra Papini, from Istituto di Tecnologie Biomediche in Pisa, and Livia Roseti, from the Istituto Ortopedico Rizzoli in Bologna. The presentation by Santini et al. is published in this same issue [3] while that of Papini et al. will appear in a forthcoming issue.
The overview of the National and European Regulations discusses lectures from Italian scientists that have elaborated the Italian guidelines and are now collaborating with the European Medicinal Evaluation Agency (London, England) to write European standards for cell therapy. The panel of Italian experts on cell therapy was established by the Italian government in 1997 and has elaborated official guidelines for gene [4] and cell [5–7] therapy, which are currently operational. These guidelines require clinical trials on cell therapy to be subjected to approval by the Health Minister and prior evaluation by experts from the Istituto Superiore di Sanità. Italian clinicians are very active in designing clinical trials in cell therapy. The Istituto has held, in only 2 years, 12 individual hearings to illustrate how the applications for approval should be prepared, and has evaluated and approved a total of 10 different protocols, most for phase I clinical trials. During the round table discussion, Roberto Lemoli, from Ospedale Sant’Orsola, Bologna, Lorenza Lazzari, from Istituto di Immunologia dei Trapianti, Milano, and Maria Grazia Roncarolo, from Ospedale San Raffaele, Milano, described the problems they have encountered in writing clinical protocols according to the criteria defined by the Italian guidelines, while Francesco Frassoni, Istituto Tumori, Genova, discussed the importance of the role of the scientific community in the process, by providing to the regulatory agency suggestions of the most scientifically appropriate criteria possible that should be included in the guidelines, in the interest of the patients.
Epithelium
In the session on the epithelium, Yann Barrandon, Michele Luca, Graziella Pellegrini, and Catherine Booth discussed the concepts of epithelial stem cells and described their clinical use to cure diseases such as full-thickness burns, vitiligo, severe deturpation, and corneal stem cell deficiency.
The session began with a review on the epidermis and its stem cells by Dr. Barrandon, a pioneer in ex vivo expansion of skin explants for transplantation [8]. The epidermis is a multilayered epithelium that survives through a self-renewing process [9–11]. The human epidermis, for example, is completely renewed every month. This process, as well as wound healing-related emergencies, is accomplished through a stem cell compartment localized in the epidermal basal layer and in the hair matrix, which, upon division, replaces its own number, generates transient amplifying cells, and gives rise to cells that differentiate into one or more specialized cell types that are eventually shed as they age [8, 9]. A consequence of this precisely organized cell hierarchy is that any attempt at using keratinocytes for permanent coverage of massive full-thickness burns requires a culture environment that allows appropriate maintenance of stem cells.
As reviewed by Dr. De Luca, it is since 1975 [12] that normal human keratinocytes have been serially propagated in vitro under defined culture conditions with the aim to replace trauma-related skin losses. It is since 1981 that in vitro expanded autologous and allogenic keratinocytes have been successfully used to reconstitute the stratified squamous epithelium of severely burned patients with proven life-saving effects [13]. In most of the cases, the graft maintains, over time, biochemical, morphological, and functional characteristics of authentic epidermis [12]. It was discussed that the great variability of clinical results present in the literature could be due to variability of ex vivo culture conditions in sustaining self-renewal of the epithelial stem cells. It was suggested that the stemness of cultured epithelial cells should be controlled by appropriate clonal assays to ensure that skin grafts obtained at different cell processing facilities maintain similar functional properties and do not loose proliferation potential during expansion in vitro.
The culture conditions for epithelial cells have improved over the years, and now they allow not only expansion of keratinocytes, but also of melanocytes and of stem cells for sebaceous glands and hair. Skin grafts with cultured cells have become, then, not only safer but the grafted skin has a progressively more natural looking appearance. For this reason, the use of cultured epithelial cells obtained from the epidermis, as well as from other lining epithelium, has broadened to include severe, although not life-threatening, applications such as stimulation of wound healing in chronic leg ulcers; reconstitution of oral mucosa affected by palatal or gingival defects; treatment of congenital urethral defects; restoration of damaged corneal surfaces, cosmetic epidermal regeneration after removal of giant nevi, scars, keloids, or tattoos [14]; and, after removal of the skin by diathermosurgery, autologous epidermal grafts in the treatment of vitiligo [15]. Furthermore, the identification that the upper region of the outer root sheath of the vibrissae in adult mice contains stem cells that respond to a morphogenetic signal to generate multiple hair follicles, sebaceous glands, and epidermis, has been used not only for the generation of better and more functional skin grafts containing appropriate melanocytes, glandular components, and hair, as discussed earlier, but also for a cell therapy approach to baldness therapy [16]. Because ex vivo expanded keratinocytes must retain stem cell properties to be functional as grafts, these cells are ideal candidates as substrates for gene therapy. Dr. De Luca also presented a clinical trial for the gene therapy of a genetic skin defect: junctional epidermolysis bulbosa [17].
Graziella Pellegrini described the clinical use of autologous fibrin-cultured limbic stem cells for the treatment of ocular disorders characterized by complete limbic stem cell deficiency. This deficiency is associated with severe ocular burns and leads to corneal opacification and visual loss. She described a new culture system that involved harvest of autologous epithelial stem cells from the limbus of the contralateral eye and their expansion on a fibrin substrate [18]. She also presented the results of a clinical trial in which these cells were transplanted in a homogeneous group of 18 limbic stem-cell-deficient patients selected on the basis of the gravity of the clinical picture and keratin expression pattern. In 78% of the patients grafted with fibrin-cultured limbic stem cells, re-epithelialization was observed within the first week, inflammation and vascularization regressed within the first 3–4 weeks, while the corneal surface became covered by a transparent, normal looking epithelium by the first month. The 12–27-month follow-up indicated that the corneal surface had become clinically and cytologically stable and the visual acuity had improved from light perception/counting fingers to 0.8–1.0 [19].
Finally, Catherine Both summarized the strategies being developed in another stem cell organized structure, the intestinal epithelial crypt. The surface of the gastrointestinal tract is lined with a simple columnar epithelium that is folded to form a number of invaginations, or crypts, that are embedded in the connective tissue. The cell organization of the crypt is reminiscent of the dermal epithelium: with the exception of the Paneth cells, the epithelial cells of the gut move upward toward the lumen as they mature. Because of this continuous upward migration, the location of a cell within the migratory stream indicates its stage in the process of maturation. Hence, differentiated, functional cells are found mainly in the villi (small intestine) or toward the top of the colonic crypt and are eventually shed into the lumen, while stem cells reside just above the crypt base in the small intestine and at the crypt base in the colon (under steady-state conditions, four to six functional stem cells per crypt [20]). As one of the most rapidly proliferating tissues in the body, the epithelium of the intestine is, together with the hematopoietic system, one of the most sensitive to chemotherapy and radiation damage. Gut stem cells cannot be identified morphologically or distinguished from other epithelial cells by a specific molecular marker, with the exception of the expression of the RNA-binding protein, Msi-1 [21]. The stem cell hierarchy is, therefore, still defined on the basis of spatial distribution within the crypt and by the level of radiosensitivity of the cells. In recent years, clonogenic cultures similar to those routinely used for bone marrow (BM) cells have been developed to quantify the number of crypt stem cells [22]. These assays have been complemented by in vivo reconstitution assays involving subcutaneous transplantation of cells in immunocompromised animals. Four to twelve weeks after transplant, an intestinal-like structure, very similar to that observed during developmental organogenesis, is generated in the skin of the transplanted animals. It is, however, still unclear whether these exogenous crypt-like structures are mono- or polyclonal in origin. The progress in understanding the mechanisms that control the cell biology of the crypt will have important future relevance, at least in the prevention of chemotherapy and radiation damage. In this regard, the possible use of keratinocyte growth factor [23], one of the factors required for the maintenance of crypt stem cells in vitro for the prevention and cure of radiation sickness, is already emerging.
Cell Therapy for Osteo- and Chondro-regeneration
During its life span, the skeleton is constantly subjected to remodeling in order to keep unaltered bone tissue biomechanical properties. Fracture healing and bone remodeling are representative processes of the regeneration capacity of bone tissue. On the contrary, articular cartilage is well known for its lack of self-repair capacity, which accounts for the particular sensitivity of joints to trauma and degenerative diseases.
Skeletal stem cell research shows great promise for the development of cell therapy for both acquired and inherited diseases when tissue repair or repopulation of a damaged organ is required. Pluripotent stem cells are, in their classical definition, capable of regenerating themselves and of undergoing multilineage differentiation. Indeed, on a functional basis, they are unique cell populations with the ability both to self-renew and to display different phenotypes in response to various microenvironmental cues.
All of these studies require an integrated view of basic cell biology, bioengineering, and medicine, together with careful ethical and regulatory considerations. Nevertheless, the area seems to be mature for raising and answering serious, and so far unresolved, questions of human health care.
In the session on bone and cartilage regeneration, three main topics were discussed: A) Are circulating stem cells able to generate mesenchymal tissues? B) Can bone marrow stromal cells be considered as real stem cells? C) What are the prospects for osteochondral repair?
Ralf Huss, from University of Munich, Germany, presented data from his laboratory on the isolation and characterization of putative mesenchymal stem cells from peripheral blood. Hematopoietic stem cells can be isolated from different sources, traditionally from the BM and presently from the peripheral blood. Recent research has revealed significant information on the plasticity of stem cells, demonstrating that it is possible to generate mesenchymal progenitor cells and even predetermined tissue precursors from hematopoietic stem cells. On the contrary, it is possible to achieve hematopoietic reconstitution with mesenchymal stem cells from different adult tissues [24, 25]. Adult stem cells with a mesenchymal phenotype can also be isolated from peripheral blood mononuclear cells and used for cellular or tissue engineering in vitro or tissue repair in vivo [25, 26], so it is possible to generate different tissue progenitor cells in vitro by applying different induction protocols and using different growth factors or cell culture conditions. Peripheral blood-derived stem cells can show, for example, an osteogenic or neural phenotype as well as stress-induced differentiation toward an endothelial cell type with neovessel formation. Nevertheless, their in vitro differentiation potential is limited, and the use of animal models to estimate the stem cell potential for tissue repair is required. Mesenchymal stem cells also contain a rare subset of active hematopoietic stem cells, known as Side Population, which can be isolated by flow cytometry due to their capacity to efflux Hoechst 33342 [27]. Although adult stem cells are mostly quiescent, they are also effective targets for gene transfer experiments using adeno-associated viruses or adenoviruses. In summary, peripheral-blood-derived mesenchymal (adult) stem cells can be effectively proposed for cell and tissue engineering in vitro and tissue repair in vivo.
Rodolfo Quarto reviewed the results from his group on BM-derived stromal cells (BMSCs). BMSCs are pluripotent cells that can be isolated from adult marrow and display, both in vitro and in vivo, the ability to give rise to different mesenchymal lineages [28, 29]. This cell population can be easily isolated from marrow and significantly expanded in vitro from relatively small samples. All together, the properties of BMSCs make them an ideal candidate for cell-based therapeutic strategies for a variety of disorders by both local and systemic applications [30, 31]. A critical issue is linked to the design of appropriate strategies for cell delivery [32]. Long-term engraftment of BMSCs after local transplant is well established. Intravenous infusion of these cells has been proposed as a means to support the hematopoiesis in BM transplant and as a vehicle for gene and cell therapy. However, it seems that this route of injection leads to engraftment of a very small proportion of BMSCs, possibly because they are unable to cross the endothelial barrier [31]. Several reports have shown that human BMSCs engraft after systemic infusion in conditioned immunodeficient mice, but the percentage of engraftment has been mostly assessed by polymerase chain reaction analysis and has never been quantitatively evaluated. It is conceivable that subcutaneous implantation not only avoids the loss due to the fact that BMSCs are not normally circulating cells, unable to cross the endothelial wall, but also lets the cells create a local microenvironment that enhances their survival [31, 32]. A system has been developed to assess the transplantability and the real productive engraftment of human BM cells in immunodeficient mice by measuring the biological responses of mice after transplant of BMSCs infected in vitro with a replication-defective retrovirus encoding the human erythropoietin gene. BMSCs have been transplanted either intravenously or subcutaneously with or without a three-dimensional (3D) scaffold in immunocompromised mice [31]. The data presented show that the same number of ex vivo expanded BMSCs that do not produce any detectable effect when transplanted by systemic infusion is capable of long-term engraftment and efficient expression of the foreign gene when locally transplanted in an artificial 3D scaffold, where they can readily create a favorable microenvironment.
Ivan Martin from the Department of Surgery, University Hospital, Basel, Switzerland, presented his results on osteochondral repair. Large osteochondral defects are associated with mechanical instability and usually need surgical intervention to prevent the development of degenerative joint disease. Ideally, large osteochondral defects should be repaired with a graft that can provide mechanical stability and allow early postoperative function under physiological loading conditions. In vivo studies have been performed to test the hypothesis that engineered cartilage could provide a tissue-like template allowing the orderly repair of very large osteochondral defects in adult rabbits. A layer of engineered cartilage was combined with an osteoconductive support to generate composites, which were grafted by press-fit into 7 x 5 x 5-mm osteochondral defects, the largest ever created in rabbits. After 6 months, the composite graft was extensively remodeled into a cartilage layer, with a thickness matching that of native tissue, and mature bone tissue that was perfectly integrated both with the overlying cartilage and the surrounding native bone. Chondrocytes, which were uniformly distributed in the engineered cartilage following its in vitro cultivation, aligned in vivo into characteristic columns, suggesting that the tissue was physiologically responding to mechanical loading. The results demonstrate that tissue-engineered cartilage can be used for the repair of osteochondral defects in rabbits and warrant extension to a human cell source and scale [33].
In order to extend the above described procedure to a clinical application, autologous cells isolated from small cartilage biopsies from patients must be seeded and cultured onto appropriate scaffolds only after being expanded in vitro, so that a sufficient cell number becomes available. However, human chondrocytes expanded in vitro and then seeded on biodegradable polymer scaffolds yielded constructs that contained little cartilaginous matrix and collapsed after a few days. This result was explained by the fact that chondrocytes during expansion in monolayers lose their differentiated phenotype and have a reduced ability to redifferentiate. Specific growth factors supplementing the culture medium during cell expansion can enhance the ability of human chondrocytes to redifferentiate and produce cartilage matrixes when transferred into a 3D environment. In fact, when human chondrocytes were expanded in the presence of three growth factors, namely, fibroblast growth factor-2, transforming growth factor-ß1, and platelet-derived growth factor-BB, they proliferated four times faster than without those factors and, most importantly, were able to generate tissues that were histologically and biochemically superior [34].
Engineered constructs based on human chondrocytes expanded in the presence of the selected factors and seeded into different types of biodegradable polymer scaffolds were not uniform, and the inner tissue phase was poor of cells and extracellular matrix. This result was explained by a reduced diffusion of nutrients and oxygen through the outer layer of extracellular matrix. The hypothesis was that increased mass transfer rates could allow generation of more uniform and functional tissues. In order to test the hypothesis, we built a tissue culture bioreactor applying dynamic loading and thus inducing fluid flow to/from the constructs. Preliminary studies demonstrated that short-term dynamic loading of chondrocyte-seeded scaffolds induced upregulation of cartilage markers (i.e., collagen type II, aggrecan, and chondromodulin) at the mRNA level. Ongoing work is aimed at a prolonged cultivation of engineered constructs under dynamic loading and the assessment of the amounts and uniformity of deposited extracellular matrix proteins.
In summary, after proving the principle that engineered cartilage could be used in conjunction with osteoconductive materials to repair large osteochondral defects, we focused our activities on the generation of cartilage tissues starting from adult human chondrocytes expanded from a small biopsy. Only an interdisciplinary approach based on the use of appropriate biochemical (i.e., regulatory molecules), structural (i.e., 3D scaffolds), and physical factors (i.e., bioreactors) will lead to the generation of functional cartilage grafts from a patient’s own cells.
Future Directions
Stem cell populations from a variety of tissues offer great promises for tissue regeneration, cell-based transplantation therapies, and the eventual development of clinically effective gene therapy protocols [35].
Researchers value stem cells as an important tool for basic research, as they are used for elucidating the mechanisms of development and cell differentiation, as vectors for permanent gene delivery, and as a basis for the development of in vitro models for pharmacology and toxicology testing. However, the current interest in stem cells springs from the prospect of using them to treat and perhaps cure a variety of diseases, particularly neoplastic or degenerative diseases [36].
Although stem cells also exist in other regenerating tissues and are considered tissue specific (neural stem cells give rise to neurons, astrocytes, and oligodendrocytes; gastrointestinal stem cells give rise to secretory, endocrine, and absorptive cells in the gut), the best-characterized stem cells are those responsible for mouse and human hematopoiesis. Hematopoietic stem cells maintain hematopoiesis throughout life and re-establish blood cell production after transplantation [37].
The hallmark of stem cells is the capacity to self-renew and to give rise to functionally mature, specialized cells; thus, they can be transplanted into tissues or organs and allow a continuous generation of mature cells in the damaged organs. Hematopoietic stem cells are multipotent, as they can generate at least eight distinct lineages of mature cells, possess a very large proliferation potential, are a rare population, and are deeply quiescent or slowly cycling in a steady-state condition.
Any stem cell population is likely to possess at least some of these properties. So far, only embryonic stem cells are thought to possess totipotentiality, the ability to give rise to all types of tissue. During development from blastocyst to embryo and fetus, the totipotent stem cell loses totipotency, which is replaced by a more narrow programming toward mesoderm, endoderm, or ectoderm, and finally single-tissue specificity.
A stronghold in our beliefs was that, in adult somatic tissues, the stem cell pool was exclusively present where constant mature cell regeneration was occurring, and therefore, certain tissues had a fixed number of mature cells, which, once damaged, could not be replaced by newly produced cells [38]. A second indisputable point was that somatic stem cells had a somewhat restricted differentiation potential (i.e., they could differentiate into several cell lineages all belonging to the same tissue). Now, our beliefs are being challenged by a number of sometimes conflicting observations that have caused debate but are also quite exciting [39]. They are widening and somehow changing our restricted ideas about the therapeutic approach to a number of diseases [40].
In the session entitled Future Directions, the issues of innovative therapy in the field of human adult stem cells were addressed. A beautiful example was presented by G. Cossu, who presented data from his group studies on myocytes. Growth and repair of skeletal muscle are normally mediated by satellite cells that surround muscle fibers. These cells divide at a slow rate to sustain both self-renewal and growth of differentiated tissue. In regenerating muscles, the number of myogenic precursors is much larger than that of resident satellite cells, implying migration or recruitment of differentiated progenitors from other sources. To assess whether BM cells can convert to myogenesis in response to certain stimuli, genetically marked BM cells were transplanted into chemically damaged muscle of immunodeficient mice or, in other experiments, immunodeficient mice were first transplanted with genetically marked mouse BM cells and, 5 weeks later, muscle regeneration was induced. It was shown that some BM cells migrate into the muscle areas of degeneration, undergo myogenic differentiation, and participate in the regeneration of previously damaged fibers [41].
The identity of these BM-derived myogenic progenitors and their origin are not known. It is possible that they originate from multipotent, mesenchymal stem cells that have been shown to also generate bone, cartilage, fat tissue, and endothelium. The existence of a BM reservoir of progenitors for muscle tissue that, in stress conditions, can supply precursors for muscle regeneration could open a new field in the development of cell-mediated replacement therapy for muscular dystrophy. However, experiments in dystrophin-deficient mice transplanted with the BM of syngenic, normal mice indicated that a BM transplant, as it is performed for hematopoietic reconstitution, for a number of reasons, cannot provide enough normal muscle tissue.
The cellular therapy for myopathies can also be addressed from a different angle, given the recent and thoroughly discussed reports on stem cell plasticity. A number of studies seem to indicate that the differentiation potentials of somatic stem cells are not restricted to the cell types derived from their tissue or germ layer of origin (neuronal stem cells giving rise to hematopoietic or muscle tissues, depending on local stimuli provided by the microenvironment; hematopoietic cells producing liver cells in mice and humans). Therefore, the possibility of transplanting adult pluripotent stem cells that can be induced to differentiate into a specific tissue, muscle in particular, could be postulated.
So, the question arises as to where adult pluripotent stem cells come from and how they can be identified. They could be only residual embryonic or fetal cells that did not disappear after birth, or rather, they could be very rare cells that have a well-defined role in adult tissue turnover.
But how do we recognize these truly pluripotent stem cells and how can we transplant them and direct them specifically in the tissue that needs to be repaired in sufficient numbers? The most accessible source of pluripotent adult stem cells are the hematopoietic tissues (BM, peripheral blood, or perhaps, cord blood?). Also, heavily vascularized tissues, such as muscle or brain, contain hematopoietic stem cells. Intriguingly, aorta-derived myogenic cells express myogenic and endothelial markers that are also expressed by satellite cells, including vascular endothelial growth factor receptor (VEGFR), which is expressed by a fraction of murine and human very primitive hematopoietic stem cells. So, besides the hemangioblast, an even more primitive meso-angioblast has also been detected in the microvessels of adult tissues. These and other observations suggest the existence of a hematoangiomyogenic stem cell [42].
In this context, the identification in adult human tissues of the hemangioblast [43], the common ancestor to both hematopoietic and endothelial tissues, was addressed by C. Peschle. Studies carried out in his laboratories have shown that VEGFR-2 (also known as KDR) expression in a very small fraction of human adult BM, peripheral blood, and cord blood CD34+ cells was important for the identification of primitive in vivo long-term repopulating hematopoietic stem cells and endothelial cells. Subsequent studies also show that, in mini-bulk cultures, CD34+ KDR+ cells generated hematopoietic and endothelial progeny and cells expressing cell markers of both cell lineages. Also, the initial CD34+ KDR+ cells could be expanded for up to 6 months, during which time they generated both a population of small cells, highly enriched for more primitive, week 12 LTC-ICs), and one of larger cells, comprising cells with hematopoietic or endothelial markers or both.
Furthermore, limiting dilution studies have demonstrated the presence of a single CD34+ KDR+ cell, which could generate both hematopoietic and endothelial colonies and also mixed hematoendothelial clones. These results suggest the presence, within the CD34+ KDR+ population, of a much smaller population of "hemangioblasts." Apparently, these cells can be propagated ex vivo; thus, much interest and hope are directed at such a bipotent cell population for future use in vivo for both gene delivery therapy or, perhaps, for tissue replacement, as additional data seem to suggest that such a cell population is endowed with the potential of differentiation into mesenchymal tissue, specifically, muscle cells [44].
The issue of ex vivo expansion of hematopoietic stem cells was addressed by Wanda Piacibello, who reported data generated from her group on human cord blood CD34+ cells. Primitive, long-term in vivo repopulating cells are abundant in cord blood; however, its limited physiological volume makes cord blood usable only for pediatric transplants. Despite the ease by which stem cells self-renew in vivo, in vitro manipulation often leads to proliferation associated with maturation, so that the initial hematopoietic stem cells lose their long-term in vivo repopulation ability.
The culture condition reported, by contrast, indicates that hematopoietic stem cell ex vivo expansion is feasible [45], as the in vivo long-term repopulation ability in the nonobese/severe combined immunodeficient (SCID) mouse model is in fact increased several-fold. Expanded cells also retain their self-renewal potential, as indicated by secondary and tertiary transplants. Some elements (e.g., telomere length measurement during ex vivo expansion) seem to indicate that these in vivo dividing primitive cells somehow retain extensive proliferation ability [46, 47].
The possibility of obtaining large numbers of transplantable, primitive hematopoietic stem cells from a stem cell source so close at hand opens the way for many opportunities for clinical applications, from allogeneic transplant in adult patients to in vivo gene delivery of therapeutic use in early-diagnosed diseases (from SCID or adenine deaminase deficiencies to thalassemia or storage diseases and hereditary myopathies).
A different, innovative approach to the therapy of liver diseases was reported by Ezio Laconi, who presented data resulting from studies of his group in Cagliari [48]. The issue of insufficient tissue replacement obtained by selective replacement of hepatocytes in chronic liver diseases caused by hepatocyte dysfunction is once again of great importance. The strategy developed by the researchers aims at a near-complete liver repopulation by transplanting isolated normal or genetically modified hepatocytes after having persistently blocked the proliferation of resident, diseased hepatocytes. Administration of retrorsine, a naturally occurring pyrrolizidine alkaloid, blocked endogenous hepatocyte growth in an animal model. Successful and extended liver replacement by transplanted isolated hepatocytes has been observed for up to 2 years, determining normalization of liver structure and function [49].
The new concept of obtaining a selective in vivo expansion of the transplanted or genetically modified normal cells by growth inhibition of resident cells is most intriguing and could find several applications, not only for the treatment of chronic liver diseases, but also for other tissue or organ diseases.
|
ACKNOWLEDGMENT |
|---|
|
TopAbstractIntroductionReferences |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionReferences |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| STEM CELLS | THE ONCOLOGIST | CME | ALPHAMED PRESS JOURNALS |
