20 results in Fetal Tissue Transplants in Medicine
4 - Ontogeny of human T- and B-cell immunity
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- By M. Elder, Department of Pediatrics, University of California, San Francisco, CA, M. S. Golbus, University of California, San Francisco,, M. J. Cowan, Department of Pediatrics, University of California, San Francisco, CA
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THE ONTOGENY OF THE HUMORAL and the cell-mediated immune responses, which are the primary mediators of successful organ and hematopoietic stem cell transplantation, is reviewed in this chapter. With the growing interest in using the fetus either as the source or as the recipient of stem cell or organ transplants, it is imperative that we understand the ontogeny of these two aspects of the immune system. Humoral immunity results from the antigenic stimulation of bonemarrow- derived or B lymphocytes and their subsequent differentiation into plasma cells capable of secreting neutralizing antibodies. Cellular immunity is the consequence of antigenic stimulation of thymusderived or T lymphocytes, which then function to kill cellular targets or to help B cells or cytotoxic T cells to respond appropriately to antigenic exposure.
Antibody- and cell-mediated immunity develop during fetal life and the ontogeny of the immune system is currently under intensive study (Figure 4.1). Hematopoietic stem cells originate in the yolk sac and within a few weeks of gestation are found in the human fetal liver. During midgestation the bone marrow becomes the primary organ of hematopoiesis. The maturation and differentiation of hematopoietic stem cells into B lymphocytes is much better understood than that of T cells; the characterization of both pathways has benefitted greatly by recent advances in molecular biological techniques that have allowed the identification of cell surface molecules and gene rearrangement events that are expressed sequentially in differentiating precursor populations.
Blymphocytes are the precursors of antibody-producing plasma cells and are responsible for the humoral immune response. Antibodies are antigen-specific immunoglobulins that are essential for host protection against bacteria and many viruses. Immunoglobulin production occurs in response to the binding of antigen to receptors on the B-cell surface membrane. This process usually requires the cooperation of helper T lymphocytes to secrete soluble factors, referred to as lymphokines, that are important for B-cell activation, proliferation and differentiation. However, antibody synthesis by some B cells does not require helper T cells, particularly those B cells producing antibodies against encapsulated organisms. Such T-cell-independent B cells are not present during gestation, do not become functional until the second year of life (Davie, 1985), and will not be discussed in detail here.
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1 - Differentiation and transplantation of embryonic cells in mammals
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- By R. G. Edwards, Churchill College, Storey's Way, Cambridge CB3 ODS, UK.
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MANY INSIGHTS ON REGENERATION by grafting fetal and adult tissue into recipients can be gained by studying the differentiation and formation of the embryo. This chapter will relate work on grafting embryonic cells to knowledge on early mammalian embryology, and especially the regulatory factors involved in tissue formation. It is not intended to be a comprehensive analysis of early embryology, and cited references to books and review should be consulted for this purpose. References to work on amphibians and insects will be made where necessary. The differentiation of the human embryo to approximately day 20 will be considered to be the end of the embryonic phase when the primary organ systems have been established.
Embryonic differentiation to the blastocyst
Formation of the blastocyst
The initial stages of embryonic growth involve a cascade of regulatory events. After sperm entry into the oocyte, the sperm chromatin forms when disulphide bonds are cleaved and nuclear proteins degraded as histones from the oocyte replace the protamines (Yanagimachi, 1988; Tesarik, 1992). Paternal and maternal pronuclei undergo DNA synthesis, the initiation of the S phase in the cell cycle being regulated by cytoplasmic factors in many species and probably in man (Laskey et al., 1989); the pronuclei persist for approximately fifteen hours in human eggs (Edwards, 1980).
The first cleavage results in two equal-sized blastomeres and one of these divides before the other; the right-angled orientation of their cleavage planes being an indication of the tight regulation of embryonic growth. Early embryos might release trophic factors, e.g. platelet activating factor (PAF) (O'Neill et al, 1989). Blastomeres of approximately equal size formed in successive cleavages are associated by microvilli; their cytoplasmic organelles differentiate; and compaction in 8-cell embryos heralds the formation of outer trophectoderm cells associated by desmosomes and tight junctions which enclose one or more inner cells. The human blastocyst initially has 32-64 cells at 4-5 days (Table 1.1), a blastocoelic cavity, an inner cell mass and large secretory-like cells adjacent to it (Figures 1.1- 1.4) (Edwards, 1980). In mice, the inner cell mass regulates the overlying polar trophectoderm which colonizes the mural trophectoderm lining the blastocoels.
6 - Transplantation of fetal haemopoietic and lymphopoietic cells in humans, with special reference to in utero transplantation
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- By J.-L. Touraine, Pavilion P, Département de Néphrologie, Medecine de Transplantation et Immunologique Clinique, Hôpitaux de Lyon, Lyon, France.
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THE TRANSPLANTATION of normal haemopoietic stem cells can cure a number of diseases in experimental animals as well as in humans. Stem cells, emerging from the yolk sac, migrate to the fetal liver and then to the bone marrow. In the latter two tissues, they develop in an appropriate microenvironment among a large variety of other cells. The bone marrow contains, in particular, T lymphocytes which developed in the thymus after this organ has been colonized by fetal liver or bone marrow prethymocytes and then returned back to the bone marrow or, to a lesser extent and only after the thirteenth week postfertilization, to the human fetal liver. Bone marrow transplantation (BMT) is a very effective treatment in many severe congenital disorders as well as in a number of inherited or acquired haematological diseases (Gatti et al, 1968; Koning et al, 1969; Thomas et al, 1975; Bortin & Rimm, 1977; Touraine et al., 1987).
Many patients have no HLA-identical donor available and the incompatible transplant is responsible for severe graft-versus-host disease (GvHD). Since GvHD is induced by the T lymphocytes which are present in the transplant and which react with host tissues (Grebe & Streilen, 1976; Korngold & Sprent, 1982), transplants from a donor who is not genotypically HLA-identical must be preceded by T cell depletion of the bone marrow. This manoeuvre reduces the incidence of GvHD but is associated with an increased risk of graft failure, incomplete reconstitution, EBV-induced lymphomas, or leukaemia relapse (O'Reilly et al, 1983; Fischer et al, 1986). It is therefore of considerable interest that fetal liver stem cells can reconstitute the haemopoietic and lymphopoietic systems of experimental animals and humans without severe GvHD, even in cases of full donor-host incompatibility (Bortin & Saltzstein, 1969; Lowenberg, 1975; Touraine, 1983;Prümmer et al, 1985;Champlinef et al., 1987; Touraine et al, 1987).
Since the pioneering work of Uphoff in 1958, much has been learned in the field of experimental and clinical fetal liver transplantation (FLT). Most of the studies have been carried out in mice (Lowenberg, 1975; Boersma, 1983). They have shown the effectiveness of fetal liver transplants in correcting the haematological and immunological consequences of lethal doses of irradiation (Uphoff, 1958; Lowenberg 1975; A. Aitouche & J.-L. Touraine, in preparation).
2 - Organogenesis and central nervous system development
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- By T. W. Sadler, University of North Carolina at Chapel Hill, Chapel Hill
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ORGANOGENESIS BEGINS in the third week of gestation (postfertilization) with the process of gastrulation and ends in the eighth week. During this six-week period, known as the time of embryogenesis, primordia for most of the organ systems are established and the conceptus becomes recognizable as a human fetus. These six weeks are also marked as the period of greatest sensitivity for induction of major congenital malformations and each organ system experiences its own peak(s) of susceptibility to insult during this time frame. By the ninth week, the conceptus enters the fetal period, which is characterized by the growth and continued differentiation of the tissues and organs that were established previously. Thus, most of the tissues and organs are established early in gestation as a result of cell-cell interactions, including inductive events. The remainder of pregnancy is then characterized by continued cell differentiation and growth.
Gastrulation
Gastrulation is the process that establishes the three germ layers, ectoderm, mesoderm, and endoderm, that are responsible for formation of all embryonic structures. Prior to this process, the embryo consists of two cell layers comprising the epiblast dorsally and the hypoblast ventrally (Figure 2.1). On the fourteenth to fifteenth day of gestation, the caudal half of the epiblast becomes marked by a shallow depression, the primitive streak. Its cephalic end is marked by a slight elevation, the primitive node (of Henson), surrounding the primitive pit. Cells of the epiblast migrate toward the streak, detach from this cell layer, and turn inward to lie between the hypoblast and remaining epiblast (Figure 2.2). The first cells to migrate displace the hypoblast and form the embryonic endoderm; later arriving cells form the mesoderm; and cells remaining in the epiblast form the ectoderm. Thus, all three germ layers and, ultimately, all tissues of the embryo are derived from the epiblast layer.
Ectoderm gives rise to the central and peripheral nervous systems; sensory epithelium of the ear, nose, and eye; subcutaneous glands, mammary glands, pituitary, and enamel of the teeth. Mesoderm provides: blood and lymph cells; endothelium of blood vessels and the heart; kidneys and gonads; cortical portion of the suprarenal glands; and the spleen.
10 - Transplantation of ovaries and testes
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- By R. G. Gosden, Department of Physiology, University Medical School, Teviot Place, Edinburgh EH8 9AG, UK.
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FEW SUBJECTS IN EXPERIMENTAL SURGERY have attracted as much public interest or professional controversy as the transplantation of testes and ovaries. At one time hailed as a cure-all for old age, clinical transplantation of gonads was eventually overtaken by advances in chemical endocrinology and transplantation biology and is now regarded mainly as one of the blind alleys of science. The legacies of the so-called rejuvenators have effectively smothered further consideration of transplantation as a potential treatment for hypogonadism.
The first authenticated record of gonadal transplantation is attributed to an eighteenth century Scottish anatomist and surgeon, John Hunter, who grafted chicken testes and ovaries to the body cavity of hosts of either the same or opposite sex. Full details of this work have not survived and it is difficult to evaluate his claims but, since he used allografts, it seems doubtful that he could have been successful. Persistent scar tissue or hypertrophy of host gonads that were incompletely extirpated may explain his claims to success. Such dangers of misinterpretation can account for many false positive findings in a later era and for much of the confusion that followed.
It is the Gottingen biologist, Berthold (1849), who should be credited with the first successful testicular transplants, since, by using autografts, he luckily avoided the risk of rejection. When he replaced the testes of capons in their own body cavity he found that the growth of comb, plumage and courting behaviour, all of which are androgen dependent, were maintained. The transplantation of ovaries was pioneered in France by Bert (1865), but several decades passed until interest in either technique became widespread.
The turn of the century signalled an explosion of interest in gonadal transplantation. One causal factor was the dawning of the new science of endocrinology and the opportunities that transplantation offered for experimentally testing hormone secretion and action. Knauer (1896) autotransplanted rabbit ovaries to the broad ligament and peritoneal cavity and obtained evidence of normal function, including ovulation and prevention of uterine atrophy. Like most of his contemporaries, he regarded allografting as potentially successful, but failed to provide any convincing evidence to support this assumption. The first investigator to transplant fetal ovarian tissue was Foa in 1900. He made the important discovery that immature organs undergo accelerated maturation in an adult environment.
Brief bibliography on various aspects of transplanting fetal 337 tissue
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13 - Law and ethics of transplanting fetal tissue
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- By J. C. Polkinghorne, Queen's College, Silver Street, Cambridge CB3 9ET, UK.
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IT is IMPORTANT to recognize that the legal and the ethical are distinct, and only partially overlapping, categories. Lying is always ethically dubious but it becomes illegal only in certain circumstances, such as when one is under oath. Moral theory holds that in certain situations there can be an ethical obligation to break unjust laws. The mechanism of written statutes and the decisions of the courts is designed to provide ascertainable answers to legal problems. Ethical judgements, on the other hand, call for acts of discernment on which there may well not be universal agreement. Not only may the principles be in dispute but also many moral decisions involve the weighing of conflicting claims (the welfare of the mother against the welfare of the fetus, for example), which different people may assess in different ways. Consensus is sometimes difficult to achieve in the moral realm. Because of this, it should not be assumed that all adherents of a particular religious tradition hold exactly similar views on all moral issues.
While there are inescapable acts of individual judgement involved in ethical decisions, nevertheless, it is desirable that conclusions should be reached in ways that result in as consistent a practice as possible. A way of achieving this in a given area of activity is to formulate a code of practice, providing general guidelines within which individual decisions are to be made. In the area of the transplantation of fetal tissue, the Code of Practice currently operative in Britain is that recommended by the Government-appointed Committee to Review the Guidance on the Research Use of Fetuses and Fetal Material (FFMC), which reported in July 1989 (FFMC, 1989). Its Code of Practice is reproduced in the Appendix.
In English law, the fetus, while in the uterus, is not a legal person. A fetus which is born and lives ex utero, even if only briefly, becomes a legal person and acquires all the rights and status thereto attached.
A fetus which dies in utero, either naturally or as the result of a therapeutic abortion, has not been a legal person and, in consequence, the provisions of the Human Tissue Act (1961) do not apply to it. There is some uncertainty about what, if any, legal requirements there are in relation to consent for the use of material derived from such a fetus.
Appendix: Code of practice on the use of fetuses and fetal material in research and treatment
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In this Code fetus means the embryo or fetus from implantation until gestation ends and, unless qualified by the words in utero, includes the fetus outside the womb.
Treatment of the fetus
1.1. Two categories of fetus are recognised:
(a) The live fetus, whether in utero or ex utero, which should be treated on principles broadly similar to those which apply to treatment and research conducted with children and adults.
(b) The dead fetus. The determination of death shall be by reference to the absence of vital functions, as indicated by the absence of spontaneous respiration and heartbeat after consideration of possibly reversible factors, such as the effects of hypothermia in the fetus, or of drugs or metabolic disorders in the mother. This determination shall be made or confirmed by a doctor responsible for the clinical management of the mother and the fetus and not involved with the subsequent unconnected use of fetal tissue.
Only tissue from the dead fetus is ethically available for use in therapy.
1.2. It is unethical to administer drugs or carry out any procedures during pregnancy with the intent of ascertaining whether or not they might harm the fetus.
1.3. In the case of nervous tissue only isolated neurones or fragments of tissue may be used for transplantation.
Contents of the uterus other than the fetus
The contents of the uterus resulting from pregnancy other than the fetus (ie the placenta, fluid and membranes) may be used for research or therapeutic purposes subject to the conditions relating to screening at section 4.5 of this Code and those relating to finance at section 7.
Separation of the supply of fetal tissue from the practice of research and therapy
3.1. The decision tq carry out an abortion must be reached without consideration of the benefits of subsequent use. The generation or termination of pregnancy to produce suitable material is unethical.
3.2. The management of the pregnancy of any mother should not be influenced by use of the fetus in research or therapy.
7 - The biology of fetal brain tissue grafts: from mouse to man
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- By H. Sauer, Department of Medical Physiology, Pettenkoferstr. 12, D-8000 Munich 2, Germany., S. B. Dunnett, Department of Experimental Psychology, Downing Street, Cambridge CB2, P. Brundin, Department of Medical Cell Research, University of Lund, Biskopsgatan
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NEURAL TRANSPLANTATION TO THE MAMMALIAN BRAIN is a field of long-standing interest to neuroscientists. Since the first reports on cortex or spinal cord grafting experiments around the turn of the century (Thompson, 1890; Shirres, 1905), the demonstration that immature brain tissue can survive (Dunn, 1917) and differentiate (Le Gros Clarke, 1940) in the adult host brain form milestones on the way to a more widespread application of neural grafting techniques since the early- and mid-seventies (Das & Altman, 1971). The finding that grafts of fetal ventral mesencephalic tissue, rich in dopamine (DA) neurons, can reverse lesion-induced functional deficits in an animal model of Parkinson's disease (Bjorklund & Stenevi, 1979; Perlow et al., 1979) has led to increased interest in neural transplantation in a wide variety of animal models of neurodegenerative diseases and other forms of neuronal or neuroendocrine dysfunction. Neural transplants of fetal brain tissue have since been investigated in animal models of Huntington's chorea (Deckel et al, 1983; Isacson et al, 1986); Alzheimer's disease (Dunnett, 1990); hypogonadotropic hypogonadism (Krieger et al, 1982; Gibson et al, 1984); diabetes insipidus (Gash & Sladek, 1984); epilepsy (Barry et al, 1987); demyelinating diseases (Lachapelle et al, 1984); cerebellar ataxia (Sotelo & Alvarado- Mallart, 1986); callosal agenesis (Smith et al, 1987); spinal cord injury (Nygrene et al., 1977; Nornes et al., 1983;Reier, 1985); cerebral ischemia and stroke (Mudrick et al., 1988; Tonder et al, 1989; Onifer & Low, 1990); cortical hypoplasia (Lee & Rabe, 1988, 1990); drug-induced prenatal brain damage (Yanai & Pick, 1988); and retinal degeneration (Turner & Blair, 1986).
The unique experimental situation created by grafting fetal brain tissue to the adult host brain has posed many fascinating questions. Which developmental characteristics does immature neural tissue display in a differentiated environment? How does the adult host brain react to the grafted immature tissue? Does this represent an unnatural situation where abnormal development and interaction occurs or may it allow for insights into the mechanisms governing neural differentiation, plasticity and regeneration? As has gradually emerged over the last fifteen years, the developmental potential of different fetal CNS tissues grafted to the adult brain is governed - at least in part - by several basic principles.
9 - The suitability of fetal and infantile donors for corneal transplantation
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- By H.J. Völker-Dieben, Diaconessenhuis, Houtlaan 55, 2334 CK Leiden, The Netherlands.
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THE NORMAL CORNEA is clear and avascular. An opaque cornea interferes with good visual acuity and such patients are candidates for a corneal transplant. Corneal transplantation is the replacement of the central 7 to 8 mm of the diseased organ. The clarity of the graft is used as the parameter for defining graft survival. However, a crystal-clear cornea with an uncorrectable refractive error does not constitute a successful transplant, since visual acuity is not restored. It is the challenge for the ophthalmic surgeon to achieve a clear cornea with curvature that is as close as possible to normal.
Factors affecting the success of corneal transplantation
Endothelial cell density
Numerous factors affect the success of corneal transplantation: i.e. selection and storage of donor corneas, surgical techniques and postoperative therapy. The importance of good donor tissue has been appreciated from the very beginning of corneal surgery. As early as 1906, Zirm stated in his rules for grafting: ‘use a human donor cornea, which should be young and healthy’ (Zirm, 1906). Corneal surgeons have traditionally preferred young donor corneas because of their high endothelial cell density.
Figures 9.1 to 9.4 show the endothelial cell densities in four donor corneas of different ages: 5400, 3500, 2800 and 1200 cells/mm2 at the age of 2 months, 2 years, 20 years and 79 years respectively. There is no regeneration of endothelial cells and the importance of the number and viability of the endothelial cells in maintaining corneal clarity is generally accepted. The high endothelial cell density in corneas of young donors provides a greater buffer against perioperative cell loss and consequently leads to better graft survival. However, the Medical Standards of the Eye Bank Association of America state that the lower age limit for donor corneal tissue is full term birth. A majority of the American Eye Banks and corneal surgeons accepts tissue from donors as young as 6 months old. For many European corneal surgeons the lower limit for acceptance is one year. Beveridge even maintained in 1972 that 'eyes of young children (under five years of age) are unsuitable, because of steep corneal curvature and lack of rigidity (Beveridge, 1972).
8 - Clinical results of transplanting fetal pancreas
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- By B. E. Tuch, Department of Medicine, University of Sydney, New South Wales 20066, Australia.
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THE IDEA OF TRANSPLANTING HUMAN FETAL PANCREAS into diabetic humans in order to normalize blood glucose levels was first seriously entertained in 1977, when the initial use of the vascularized pancreas (Sutherland, 1981) and the islet (Najarian et al,1975) proved to be unsuccessful. What prompted the move in this direction were the elegant studies of Brown and his group in California, demonstrating that the rat fetal pancreas, when isografted at 17 days gestation into an adult diabetic recipient, was capable of normalizing the blood glucose levels within 4-6 weeks of transplantation (Brown et al, 1974). Subsequent studies by Mandel in Melbourne, Australia, showed that the fetal mouse pancreas behaved in a similar manner when isografted (Mandel, 1984). Several years later, in 1985, the human fetal pancreas was shown to be capable of reversing diabetes when xenografted in the immunoincompetent athymic, or nude, mouse (Tuch et al, 1985b). This and other studies demonstrated that human fetal pancreatic explants, when grafted into these animals, reversed diabetes within one to three months (Hullett et al, 1987; Tuch et al, 19886) (Figure 8.1). The apparent universal potential of grafted fetal pancreas to reverse diabetes gained further support with the demonstration, in 1988, of the successful normalization of blood glucose levels in the athymic mouse by explants of pig fetal pancreas (Walthall et al, 1988) and, in 1990, proislets, or islet-like cell clusters, of this tissue (Simeonovic et al, 1990; Korsgren et al, 1991).
In the Western world, human fetal pancreas is obtained from termination of pregnancies performed up to the twenty fourth week of gestation (Jovanovic-Peterson et al, 1988). In some countries, such as Australia, the guidelines are stricter, prohibiting the termination of pregnancies after 20 weeks (National Health & Medical Research Council of Australia Medical Research Ethics Committee, 1983). In the People's Republic of China, where there is a policy of one child per family, termination, which is an accepted form of contraception, may be performed at a relatively late stage of pregnancy, thus making it possible to utilize pancreas obtained in the third trimester (Hu et al, 1985).
3 - Experimental human hematopoiesis in immunodeficient SCID mice engrafted with fetal blood-forming organs
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- By B. Péault, SyStemix Inc, 3400 W. Bayshore Road, Palo Alto, California 94303, USA., R. Namikawa, SyStemix Inc, 3400 W. Bayshore Road, Palo Alto, California 94303, USA., J. Krowka, SyStemix Inc, 3400 W. Bayshore Road, Palo Alto, California 94303, USA., J. M. McCune, SyStemix Inc, 3400 W. Bayshore Road, Palo Alto, California 94303, USA.
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THE MATURE HEMATOPOIETIC SYSTEM retains conspicuous embryonic features: extensive stem cell production, migration and differentiation ensure in the adult a permanent supply of myeloid and lymphoid elements. This enormous capacity for regeneration provides a unique opportunity for the investigator who can controllably reconstitute animals whose blood system has been ablated with cytotoxic drugs or ionizing radiation. Such experimental approaches in the mouse have been seminal in the study of stem cell biology, hematopoietic differentiation in blood-forming organs and 'self restriction of T lymphocyte function. Similar studies in man are, of course, deontologically unacceptable. Therefore, our understanding of human blood cell physiology still relies largely on animal paradigms or on the evaluation of in vitro cultured cells.
These approaches are of uncertain relevance and useful only in limited areas of investigation. Growing intact human organ pieces in a laboratory animal could obviously offer a key for the experimental study of living tissues in man. Transplantation is ideally achieved between histocompatible, syngeneic individuals, but xenogeneic chimeras can be constructed if the recipient is unable to mount a graftrejecting immune response. This condition can be met if the host is either an embryo, whose immune system has not developed yet (see LeDouarin (1978) for a review), or a congenitally immunodeficient adult. For example, human fetal tissues such as brain and pancreas have been implanted into athymic, partially immunodeficient rodents (Tuch et al, 1984; Stromberg et al., 1989). In a further step, lymphocyte-free mice, affected by the SCID (severe combined immunodeficiency) mutation, were used as recipients for human fetal bloodforming tissues (McCune et al, 1988) or isolated hematopoietic cells (Mosier et al, 1988).
Homozygous C.B.-17 scid/scid mutants, referred to hereafter as SCID, lack functional T and B lymphocytes as a consequence of defective rearrangements in antigen receptor genes (Bosma et al, 1983; Schuler et al, 1986). The prediction that human fetal tissue and SCID mice should be tolerant of each other was realized beyond expectations: fetal thymus was observed not only to survive in the mouse but to grow, differentiate and sustain hematopoietic activity. This discovery prompted attempts to approximate the cycle of human lymphopoiesis in the SCID mouse more closely by supplying the grafted thymus with hematopoietic precursor cells.
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5 - The procurement of human fetal tissues for clinical transplantation. Practice and problems
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- By L. Wong, MRC Tissue Bank, Royal Marsden Hospital, Fulham Road, London SW3 6JJ, UK.
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THERE HAS BEEN A RECENT RENEWED INTEREST in the USe of human fetal tissues for clinical transplantation. At present this use is small in comparison to use in research. However, provided that the ethical and moral concerns of society can be resolved, the potential for medical benefit is considerable.
The procurement of human fetal tissues for transplantation and research are essentially similar, except that, for the former, steps should be taken to ensure the safety of the tissues for the recipient. Only the principles of procurement can be covered in this general article which is largely based on fourteen years of the author's practical experience in acting as an intermediary in the procurement of and working with human fetal tissues. It must be emphasized that any views expressed are those of the author. The ethical and legal requirements discussed are those which are currently in force within the United Kingdom at the time of writing.
For the purposes of this article the definition of human fetal tissue transplantation is taken as the transplantation of tissues or cells from a human fetus into a human recipient for therapeutic purposes. Although pregnancy may be regarded as a form of naturally occurring embryo transplant, the intrauterine implantation of human embryos derived from in vitro fertilization techniques is not included under this definition. With the possible exceptions of human fetal donor thymus transplantation and adult donor bone marrow transplantation, the essential difference between adult organ and fetal tissue transplantation is that adult organ transplantation aims to replace a nonfunctional organ with a like but fully functional mature organ, whereas fetal tissue transplantation aims to transplant immature tissues or cells which have the potential to grow and mature within the host. There is a lack of consistency in the definitions of embryo and fetus that are used in the embryological literature and those used in ethical and legal documents. However, since the use of fetal tissues in clinical transplantation in the United Kingdom comes within the remit of the recommendations of the Polkinghorne Report (1989), the definition of fetus used in this article is taken from that Report and covers the period of development from implantation until term.
List of contributors
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12 - The low temperature preservation of fetal cells
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- By M. J. Ashwood-Smith, Department of Biology, University of Victoria, PO Box 1700, Victoria, British Columbia, Canada.
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A BANK OF CELLS AND TISSUES that are conveniently available for experimentalists and clinicians is clearly desirable. Storage at standard refrigerator temperatures (4°C) cannot be used for most cells for periods longer than a few hours without serious imbalances in ion concentrations, protein conformation and, in particular, cytoskeletal structure. Microtubules are very temperature sensitive and effects may or may not be reversible, depending on the time and temperature of exposure. Although most of these observations were made with mouse oocytes (Johnson & Pickering, 1987) similar changes in microtubules can be seen with tissue culture cells (M. A. Hammer & M. J. Ashwood- Smith, unpublished observations).
Cryopreservation at temperatures that are lower than — 130°C permits the banking of most individual cells of biological and medical interest (Ashwood-Smith & Farrant, 1980) and has, in recent years, had an important impact on animal breeding and the clinical practice of IVF with the successful preservation of embryos (CIBA Foundation, 1977; Ashwood-Smith, 1986). Before discussing the progress made in fetal tissue cryopreservation a brief overview of cryobiological principles is apposite.
Basic cryobiology
Cells and tissues must be stored at temperatures lower than — 130 °C to ensure complete stability, otherwise some reactions and the recrystallization of ice can still occur. Cells may, however, be kept at — 70 °C to -80°C for several months without undue damage. Cryopreserved cells in liquid nitrogen (—196 °C) can be stored for many years. However, ten years is the maximum recommended time for human embryos, although this time limit has no scientific basis, since the effects of ionizing radiation (both cosmic and from radioactive isotopes) are so small as to constitute no hazard. A period of 32000 years of storage at -196 °C would lead to an accumulated dose of approximately 3.5 Gy (350 rads) which is the LD50 for most mammalian cells and over a ten year period in the frozen state an embryo would be exposed to an accumulated and fractionated dose of approximately 0.001 Gy (0.1 rads). These calculations take into account the combined radioprotective effect of temperature and the cryoprotectant (glycerol or dimethyl sulphoxide). Upon cellular resurrection from the frozen state, accumulated damage to the DNA is manifested, since the normal DNA repair enzymes cannot function at cryogenic temperatures (Ashwood-Smith & Grant, 1977).
Preface
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- By R. G. Edwards, Churchill College Cambridge
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- Fetal Tissue Transplants in Medicine
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- 05 November 1992, pp xi-xii
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Summary
THE SUBJECT OF THIS BOOK has held my interest for many years. It was greatly stimulated in 1962, in John Paul's laboratory in Glasgow University, as we saw various types of tissue differentiate in outgrowths of stem cells from rabbit blastocysts. The implications of the work led me to the concept of using human blastocysts to obtain tissue cultures of stem cells for human grafting, and became one of the driving forces leading me to undertake human in vitro fertilization. Success has not come easily in obtaining these cell outgrowths from human embryos, although there are now promising leads in this direction. I trust that my dedication to the clinical application of this field is reflected in the pages of this book, and that it will stimulate more studies on grafting fetal tissue. The book focusses on one of the many clinical uses of fetal tissue: for grafting into sick fetuses, children and adults of all ages. Fetal tissue has various known benefits. Tissues that are difficult to obtain from adults are more readily available from fetuses. Fetal tissue has already been widely applied in some forms of grafting, as in the reconstitution of the haemopoietic system in adults, even though tissue from adult donors who are closely related to the recipient is used more often. Large numbers of dynamic and adaptable fetal cells are available, capable of a sustained and widespread colonization of many tissues in both fetal and adult recipients. Fetal tissue might also offer advantages in avoiding graft rejection in a manner not matched by adult donor tissues. Finally, the astonishing ability of embryonic stem cells, sometimes modified genetically, to colonize preimplantation embryos may be of considerable value if it could be adapted for grafting into fetuses or adults. The book is designed to progress through modern knowledge, beginning with chapters by Sadler and myself which provide the framework of embryonic differentiation and organogenesis in man. Successive contributions on the ontogeny of human immunity by Elder and co-authors, and on the value of experimental animals as experimental models by Peault and his colleagues provide data that is essential to investigate the value of fetal tissue in grafting. Most of the remaining chapters of the book describe specific clinical advantages of fetal tissue in the recolonization of particular recipient organs.
Index
- Edited by Robert G. Edwards
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- Book:
- Fetal Tissue Transplants in Medicine
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- 05 November 1992, pp 347-352
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11 - Cell grafting and gene therapy in metabolic diseases
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- By M. Adinolfi, Division of Medical and Molecular Genetics, Paediatric Research Unit, Prince Philip Research Laboratories,
- Edited by Robert G. Edwards
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- Fetal Tissue Transplants in Medicine
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- 05 November 1992, pp 281-298
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Summary
THE LONG-TERM PROSPECTS FOR A SUCCESSFUL CORRECTION of inherited lysosomal enzymatic deficiencies by the transplantation of enzymatically normal cells depend on a series of interconnected factors (Nadler, 1980; Desnick & Grabowski, 1981). The most important condition is the availability of donor cells capable of producing and secreting specific enzymes, whilst not expressing histocompatibility antigens which may induce an immunological rejection. The donor cells should also be immunologically incompetent and, consequently, unable to trigger graft-versus-host (GVH) reactions. Another essential factor is that the lysosomal enzyme released by the donor cells should be taken up by the enzymatically deficient cells of the host via specific receptors.
In this chapter, I will analyze these and other requirements for achieving a successful transplant and summarize the results of studies performed to correct selected enzymatic deficiencies, particularly in patients with mucopolysaccharidoses (MPS), by transplanting normal fetal fibroblasts or amniotic epithelial cells. Alternative strategies, such as the use of somatic gene therapy, will be outlined briefly in view of their possible future applications.
Grafting fibroblasts or amniotic epithelial cells
The rationale for choosing fibroblasts or amniotic epithelial cells
The willingness to risk failure is an essential component of most successful initiatives. The unwillingness to face the risk of failure - or an excessive zeal to avoid all risks - is, in the end an acceptance of … abdication’ (Shapiro, 1990, p. 609).
Perhaps these words best summarize the hesitations and doubts which accompanied some of the early attempts performed in order to correct selected lysosomal enzymatic deficiencies by transplanting normal fibroblasts or amniotic epithelial cells. Both types of cells were known to have intrinsic disadvantages when compared to bone marrow cells, which divide and spread rapidly - ultimately producing larger quantities of a specific enzyme - but may be rejected, if HLA incompatible, or induce GVH reactions (Hirschhorn, 1980; Parkman, 1986). On the other hand, the most important incentive for grafting fibroblasts or amniotic epithelial cells was the knowledge that these cells were not going to be harmful to the transplanted patient, since they lack, or produce only small quantities of, major histocompatibility complex (MHC) antigens and do not transform readily into cancer cells.
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