Perpetuation of the species requires that a significant proportion of a population avoid premature death in order to achieve reproductive maturity. This requires a homeostatic mechanism to counter entropic processes and maintain the integrity of tissues. That mechanism is regeneration. Tissues that undergo cellular turnover, such as blood and epithelia, regenerate continually, a process called maintenance regeneration. These tissues, as well as some others, also regenerate on a larger scale when damaged, a process called injury-induced regeneration. Richard J. Goss has concisely summed up the relationship between regeneration, life and death in the following words. “If there were no regeneration there could be no life. If everything regenerated there would be no death. All organisms exist between these two extremes. Other things being equal, they tend toward the latter end of the spectrum, never quite achieving immortality because this would be incompatible with reproduction” (Goss, 1969).
Regeneration maintains or restores the original architecture of a tissue by recapitulating part of its original embryonic development. Nature, however, has provided us with another, much more common, mechanism for injury-induced repair. This mechanism is fibrosis, which patches a wound with scar tissue, lowering its functional capacity. Fibrosis is the result of an inflammatory response that produces a fibroblastic granulation tissue that is then remodeled into a collagenous scar. Mammalian tissues that do not regenerate spontaneously are repaired by fibrosis. Even tissues capable of regeneration may repair by fibrosis if they suffer wounds of a size that exceeds their regenerative capacity. Furthermore, the ability to regenerate can be destroyed or neutralized by chronic degenerative diseases, again resulting in fibrotic repair. Prominent examples of tissues that undergo fibrotic repair after damage are articular cartilage, the dermis of the skin, the pancreas, the spinal cord and most regions of the brain, the neural retina and lens of the eye, cardiac muscle, lung, and kidney glomerulus.
The cost of tissue damage due to regenerative incompetence is enormous in terms of health care (estimated to exceed $400 billion in the United States alone), lost economic productivity, diminished quality of life, and premature death. The health care costs of spinal cord injuries, which are some of the most devastating known, exceed $8 billion per year and $1.5 million per patient over a lifetime in the United States. Diabetes, heart, liver, and renal failure, emphysema, macular degeneration and other retinal diseases, diseases of the central nervous system such as Parkinson’s, Huntington’s, and Alzheimer’s, arthritis, burns, and various sports injuries that damage ligaments, tendons, and joints are other major contributors to these fiscal and human costs. Thus medical science seeks ways not only to prevent and cure underlying disease, but to restore the structure and function of damaged tissues and organs.
There is currently great excitement over the possibility of replacing damaged body parts through the new field of regenerative medicine. Potential strategies of regenerative medicine include stem cell transplantation, implantation of bioartificial tissues synthesized in the laboratory, and the induction of regeneration from the body’s own cells by rendering the injury environment and/or responding cells regeneration-competent. What has been forgotten in the excitement, however, is that regenerative medicine will not become a reality without a fundamental understanding of the mechanisms of regeneration, driven by advances in molecular, cell and developmental biology, and information science and technology. This understanding is far from complete. Thus I call this new field of tissue restoration “regenerative biology and medicine” to emphasize that understanding the biology of regeneration is prerequisite to establishing a regenerative medicine. The objective of regenerative biology is to define the factors that lead to a regenerative response and how these factors differ in a fibrotic response to injury. Regenerative medicine then seeks to apply this knowledge to devise therapies that will stimulate the functional regeneration of damaged human tissues that do not regenerate spontaneously, or whose regenerative capacity has been compromised.
A Brief History of Regenerative Biology and Medicine
Our current knowledge of repair by fibrosis and regeneration has a long and fascinating history. Imprints of hands with missing fingers on the walls of Paleolithic caves have been interpreted as examples of amputations (Goss, 1991). Severe injuries such as penetrating wounds, multiple fractures, spinal cord compression, or damage to the eyes must have occurred frequently in prehistoric times, often with high morbidity and mortality. The facilitation of wound healing and surgical intervention was a central focus in the medicine of ancient Sumerian, Egyptian, Chinese, Indian, and Incan civilizations (Majno, 1975; Brown, 1992; Falabella, 1998). The cleansing and debridement of wounds was a common practice in ancient cultures and many different vegetable and mineral concoctions were applied topically to treat wounds. Honey and wine were used as antiseptics by the ancient Chinese and Egyptians. For 2000 years the Chinese have used a bread mold to treat minor burns (Majno, 1975; Fu et al, 2001). Trephination to relieve intracranial pressure was performed by the Incas in the treatment of head wounds (Majno, 1975). A thousand years ago, the Indian physician, Sushruta, described the use of autogeneic skin transplants to reconstruct severed noses and ears (Majno, 1975).
The Greek and Roman physicians Hippocrates (460-370 BC), Celsus (25 BC – 50 AD), and Galen (130 - 201 AD) contributed greatly to the development of medicine, including the treatment of wounds (Brown, 1992). Celsus described the four cardinal signs of wound inflammation, “rubor et tumor cum calore et dolore” (redness and swelling with heat and pain). Part of Galen’s career was spent caring for injured gladiators, giving him extensive experience in treating wounds to many parts of the body. Galen compiled virtually all that was known about anatomy, physiology, and medical treatment (much of it erroneous) into at least 35 volumes. When the Western Roman Empire collapsed near the end of the 5th century AD, Galen’s texts were translated into Arabic, which was the language of medicine in the Eastern Roman Empire, and later from Arabic into Latin. These texts were the dominant guides for medical practice to the end of the Middle Ages.
A creative resurgence in biology and medicine took place from the 14th to the 17th centuries. A major contribution was the detailed and accurate anatomical descriptions of embryonic and adult structure made by Da Vinci (1452-1519), Vesalius (1514-1564), Paracelsus (1493-1541), Fallopius (1523-1562), and Fabricius (1537-1614). Descartes (1596-1650) and Borelli (1608-1679) wrote important texts on physiology, and Harvey (1578-1657) produced his famous treatises on the circulation of the blood and the reproduction of animals. These studies laid the foundation for our later understanding of form and function. The surgeon Guy de Chruliac (1300-1370) published “Wounds and Fractures” in 1363, a book that details many kinds of wounds and how to treat them. Wilhelm Fabry (1560-1634) described nearly 70 topically applied formulations for treatment of wounds, many of which have been recently re-examined and found to have true therapeutic value (Kirkpatrick et al, 1996).
The 17th century saw the introduction of the scientific method as a powerful way of answering questions about how things work, changing our view of the world forever (Van Doren, 1991). The physical laws relating forces and motion were discovered by Galileo (1564-1627), Kepler (1571-1630) and Newton (1642-1727). These discoveries and the mathematical ideas of Descarte established the philosophy of mechanism, which held that all natural phenomena have causes that can be explained by deducible mathematical laws of physics. The establishment of mechanism in the biological world was aided by the invention, in the early 1600s, of the compound microscope, which made it possible to view biological structure in greater detail than ever before and which led to the development, in the 18th century, of the science of microscopic anatomy. Studies with the microscope toppled the dogma of organismic preformation, which held that reproduction took place simply by the growth of a tiny predelineated adult in the egg, by showing that new beings developed in a continuum of epigenetic steps that built form out of amorphous substance. Simultaneously, the comparative anatomist John Hunter (1728-1793) turned the microscope on healing skin wounds and discovered granulation tissue and the transitional role it played in scar tissue formation (Brown, 1992).
Primitive people were likely aware of the ability of certain food animals, such as male deer and elk, to regenerate antlers, and crayfish or lobsters to regenerate limbs. They were also undoubtedly cognizant of the fact that hair and
nails grow continually. The regeneration of organs and appendages is a theme found in the ancient Greek mythologies of the Hydra and of Prometheus, recounted by Homer and Hesiod (Dinsmore, 1998). The Second Labor of Hercules was to slay the nine-headed Hydra, feared for its remarkable power of regenerating two heads for every one sliced off. The Titan god Prometheus made the mistake of offending the supreme god, Zeus, by stealing fire from Olympus for the benefit of humankind. As punishment for this deed, Zeus had him chained him to a rock, where each day his liver was eaten by an eagle, only to regenerate at night. This cycle of hepatectomy and regeneration would have been eternal had not Prometheus been rescued by Hercules. Accounts of regeneration of severed human arms and legs are also woven into the superstitions and descriptions of miracles in the Middle Ages (Goss, 1991).
Regeneration became a focus of systematic scientific investigation in the 18th century. Abraham Trembly performed detailed experiments on the regeneration of hydra that made a deep impression on the biologists of the time (Lenhoff and Lenhoff, 1991), while Reaumer and Spallanzani reported their observations on the regeneration of limbs in crustaceans and newts, respectively (Skinner and Cook, 1991; Dinsmore, 1991).
The 19th century saw numerous medical and surgical advances that improved the prospects of recovery from serious injury and disease (Allen, 1977). The development of ether anesthesia made pain-free surgery possible and thus increased the types of wounds that could be made in the human body for the purpose of surgical treatment. Surgery, however, also increased the possibility of death by systemic bacterial infection. Joseph Lister, a student of Pasteur, introduced the use of dressings soaked in carbolic acid and strict hygenic measures in hospitals to combat sepsis after surgical operations (Brown, 1992). Skin grafting, described by Sushruta many centuries earlier (Majno, 1975), was revived by 19th century surgeons.
By far the most fundamental and significant development of the 19th century for biology and medicine was the rise of materialistic biology, driven by microscopic observation and the conviction that life processes are explainable solely in terms of chemical reactions dictated by the properties of material substances as opposed to immaterial vital forces, which up until that time were used to explain the special properties of life (Coleman, 1977). This materialism was a natural consequence of mechanistic philosophy and experiments in chemistry begun by Lavoisier in the 18th century, which showed that life depended on chemical reactions that were reproducible in the laboratory. A key conceptual advance was the formulation of the cell theory by Schleiden and Schwann in 1838-1839 and the later microscopic observations of Virchow, Remak and others, which led to the idea that cells are the fundamental units that carry out the chemical reactions of life and that new cells are created by the division of existing cells.
Studies on limb development and regeneration in the latter part of the 19th and the early part of the 20th centuries made major contributions to the understanding of development. Prior to the 20th century, regeneration had been explained by the growth of preformed copies of tissues and appendages residing within the originals, driven by vital forces. Now regeneration was recognized as a regulative process evoked in the remaining part that restored the whole. A major research aim of Thomas Hunt Morgan (1866-1945), before he turned to genetics, was to explain regeneration in terms of chemical and physical principles. Over a century later, we are still in the process of formulating this explanation.
The 20th century saw an unparalleled explosion of biological and medical knowledge. Major advances were the discovery and production of antibiotics, an understanding of the immune system that revealed the antigenic differences between “self” and “non-self”, and the development of highly sophisticated imaging and surgical technologies. These advances, coupled with advances in engineering and materials science and the development of immunosuppressive drugs, have given us the ability to transfuse blood and replace damaged and dysfunctional tissues and organs through tissue and organ transplants and implants of bionic devices.
Without question, however, the most fundamental and far-reaching event of 20th century biology was the discovery, in mid-century, that DNA is the hereditary material (Avery et al, 1944; Hershey and Chase, 1952) and that it has a helical structure consisting of two deoxyribose sugar –phosphate backbones held together by complementary base pairs, adenine to thymine and guanine to cytosine (Watson and Crick, 1953). The power of this structure to explain how the genetic material is replicated and mutates, how information for protein structure is encoded in it and expressed, was enormous, and has led to exponential advances in our knowledge of cell biology, embryonic development and evolution. In the process, the age-old dream of being able to regenerate tissues and organs that do not regenerate spontaneously has re-emerged.
The Biology of Regeneration
Embryonic Stem Cells Give Rise to all Adult Cells
All of the trillions of cells in the adult body are derived from less than 100 embryonic stem cells (ESCs) of the pre-implantation blastocyst. ESC cultures have been established from the early embryos of fish, birds, and a variety of mammals (Smith, 2001). Human ESC cultures have been established from unused frozen blastocysts produced by in vitro fertilization in assisted reproduction facilities, and from primordial germ cells of 5-9 week embryos (Thomson et al, 1998; Shamblott et al, 1998). These cells express stage-specific embryonic antigens (SSEA-3 and 4, TRA-1-60 and 81), alkaline phosphatase, and high levels of telomerase. ESCs, whether freshly isolated, or cultured for long periods of time, are pluripotent, capable of giving rise to any of the more than 200 differentiated cell types of the body. This pluripotency has been unequivocally demonstrated in vivo by injecting ESCs into host blastocysts, where they make contributions to all tissues to form a chimeric embryo (Smith, 2001). In mammals, all the cells of the blastocyst inner cell mass are pluripotent. Prior to implantation, the pluripotent cells become restricted to the epiblast of the embryo. Epiblast cells are self-renewing and pluripotent up to gastrulation, when they begin progressive differentiation into the three germ layers and their derivatives (Smith, 2001).
The acquisition and maintenance of pluripotency of mouse ESCs requires the cooperation of leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP). BMP induces the expression of inhibitor of differentiation (Id) genes via Smad transcription factors (Ying et al, 2003). LIF activates STAT-3. In turn, STAT-3 activates the homeodomain transcription factors OCT4 (Smith, 2001), SOX2 (Avilion et al 2003) and Fox D3 (Hanna et al, 2002). Recently, a LIF/STAT3-independent transcription factor, Nanog, has been discovered that is also essential for mouse and human ESC pluripotency and self-renewal (Mitsui et al, 2003; Chambers et al, 2003). Nanog is downregulated as cells exit from the primitive streak to become mesoderm, while OCT4 is still expressed. As development proceeds, however, all the pluripotency genes are downregulated, except in the germ cells (Hart et al, 2004).
Embryonic stem cells give rise to the three germ layers of the embryo - ectoderm, endoderm and mesoderm – from which differentiated adult tissues emerge. As ontogenesis proceeds, the cells of these germ layers become progressively more diversified. Ultimately, diversification proceeds to the determination of progenitor cells for each terminal cell phenotype. The progenitor cells divide to make more progenitor cells (amplification), which then differentiate into the functional cells.
Regeneration Takes Place at All Levels of Biological Organization
All organisms regenerate, though the degree of regenerative ability varies among species and with level of biological organization within the individual organism (Goss, 1969). For example, a single carrot cell can regenerate a whole carrot (Steward, 1970). Some species, such as planaria and hydra, can regenerate whole organisms from fragments of the body (Goss, 1969; Baguna, 1998; Sanchez-Alvarado, 2000). Certain amphibians can regenerate complex structures such as limbs and tails, as well as many other tissues. Compared to these life forms, the regenerative capacity of mammals, including humans, is limited, but no less vital.
Within individual organisms, regeneration takes place from the molecular to the tissue levels of biological organization.
1. Molecular level
On the molecular level, regeneration is ubiquitous. All cells can adjust the balance of protein synthesis and degradation in response to biochemical or mechanical load. For example, cardiomyocytes replace most of their molecules over the course of two weeks and adjust their rate of protein synthesis upwards under a sustained increase in blood pressure, becoming hypertrophied (Gevers, 1984).
2. Single cell level
On the single cell level, regeneration is more restricted. The free-living unicellular protozoans can regenerate complete cells after removal of large fragments as long as nuclear material is present in the remaining part (Goss, 1969). For example, as little as 1/ 80 of an amoeba is capable of reconstituting a complete amoeba (Vorontsova and Liosner, 1960). In vertebrates, the axons of sensory and motor nerves are capable of regeneration in vivo after crush or transection, provided that the endoneurial tubes that encase them remain intact and in register at the site of injury (Yannis, 2001). The ends of the axons on the proximal side of the injury are sealed off and their distal part degenerates. The sealed proximal segment of the axon then sprouts an advancing growth cone, behind which the axon extends through the Schwann cell tube to make new synapses with target skin and muscle (Griffin and Hoffman, 1993; Yannas 2001).
3. Tissue level
There are three prerequisites for regeneration at the tissue level. First, tissues must contain mitotically-competent cells; that is, cells that have the receptors and signal transduction pathways to respond to a regeneration-permissive environment. Second, the injury environment and the systemic environment of the tissue must contain the necessary signals to promote the proliferation and differentiation of these cells in an organized way. Third, factors inhibitory to regeneration must be absent from the injury environment, suppressed, or neutralized. Both fibrotic and regenerative injury environments are favorable to the regeneration of blood vessels, which are essential to nourish either fibrotic or regenerated tissue. Blood, epithelia, hair, and nails are examples of mammalian tissues that have mitotically-competent cells that engage in maintenance or injury-induced regeneration. Bone, skeletal muscle, liver, small blood vessels, adrenal cortex, and kidney epithelium also contain mitotically-competent cells that regenerate new tissue in response to damage.
There are three mechanisms of tissue regeneration in vertebrates: compensatory hyperplasia, activation of reserve adult stem cells, and dedifferentiation of mature cells. Table 1 lists some regenerating tissues in vertebrates according to the mechanism by which they regenerate.
Compensatory hyperplasia
Compensatory hyperplasia is the proliferation of differentiated cells to regenerate new tissue. The differentiated cells are released from their ECM or syncytial complex and divide while maintaining all or some of their differentiated functions. The classic example of regeneration by compensatory hyperplasia is the liver (Michelopoulos and DeFrances, 1997). After partial hepatectomy, the hepatocytes of the liver, as well as its non-parencymal cell types (Kupffer, Ito, bile duct epithelial, and fenestrated epithelial cells) divide while performing their functions of glucose regulation, synthesis of blood proteins, secretion of bile, and drug metabolism, until the original mass of the liver is restored (Trembly and Steer, 1998). Other tissues that may regenerate by compensatory hyperplasia are the β-cells of pancreatic islets (Dor et al, 2004), blood vessels and newt cardiac muscle (Bettencourt-Diaz et al, 2003).
Regeneration via reserve adult stem cells
Late in embryonic or fetal life, subsets of lineage-restricted, but not fully differentiated, cells are set aside as reserve adult stem cells (ASCs). These cells may reside within a tissue, or circulate in the blood. Some are used for juvenile growth after birth or hatching and others for regeneration throughout life (Fuchs and Segre, 2000; Weissman, 2000). ASCs are self-renewing and typically divide asymmetrically to give rise to a cell with a more restricted lineage and another stem cell. They have varying degrees of developmental potential, depending on the tissue they serve. Regeneration via reserve ASCs is the most common avenue of tissue regeneration in multicellular organisms, including planarians, which regenerate via stem cells called neoblasts (Baguna, 1998; Sanchez-Alvarado 2000).
Ectodermal and endodermal derivatives regenerate from epithelial ASCs that reside in the basal layer of the epithelium. Examples are skin epidermis, hair follicles, the epithelium of the respiratory and digestive tracts, limited parts of the central nervous system and acoustic sensory hair cells (Jensen et al, 1999; Potten, 1997; Slack, 2000; Gage, 2000; Roberson et al, 1995). The liver also contains a population of stem cells that are activated when the liver is damaged beyond the capacity for regeneration by differentiated liver cells (Michaelopolous and DeFrances, 1997).
Mesodermal derivatives, such as skeletal muscle, bone and blood regenerate from non-epithelial hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and muscle stem cells (satellite cells) associated with the tissue or residing in the bone marrow (Hansen-Smith and Carlson, 1979; Ham and Cormack, 1978).
Reserve ASCs normally differentiate into a range of phenotypes within their lineage, called their prospective significance, or fate. For example, hematopoietic stem cells, which reside in the bone marrow, are multipotential and give rise to erythrocytes, myeloid cells and the several cell types of the immune system. Others, such as liver stem cells, are bipotent and give rise only to hepatocytes and bile duct cells. Still others, such as epidermal stem cells, appear to be unipotent, giving rise only to one cell type, in this case keratinocytes.
Different adult stem cell populations are characterized by molecular phenotypes that are both overlapping and distinct. A comparative strategy has been used to define a common set of genes for “stemness” in ESCs and ASCs (Ramalho-Santos et al, 2002; Ivanova et al, 2002). Transcriptional profiles of mouse ESCs, neural stem cells (NSCs, HSCs and differentiated cells from the lateral ventricles of the brain and the main population of bone marrow were compared by hybridizing DNA microarrays containing several thousand genes with mRNAs from each set of cells. Bioinformatic analysis was used to identify transcripts absent in differentiated cells but present in stem cells and to assign transcripts enriched in stem cells to functional categories. Both studies found a common set of 216 (Ramalho-Santos et al, 2002) and 283 (Ivanova et al, 2002) genes enriched in all three sets of stem cells that appear to define core “stemness”. These genes encode proteins for (1) various aspects of signaling (JAK/STAT transducers and activators of transcription and Notch signaling, ability to sense growth hormone and thrombin, and interaction of cell adhesion molecules with ECM); (2) entrance and progression through the cell cycle; (3) high resistance to stress, with upregulation of DNA repair, protein folding, ubiquitin system, and detoxifier systems; (4) transcriptional regulation, including chromatin remodeling; and (5) translational regulation. More than 50% of the commonly enriched genes were expressed sequence tags (ESTs), which represent unidentified genes. Otherwise, the gene activity of each set of stem cells is distinct from one another. HSCs are more similar to the main bone marrow population than to ESCs or NSCs, but NSCs are more similar to ESCs than to HSCs or differentiated cells (Ramalho-Santos et al, 2002).
All ASCs reside in environmental niches that maintain their stem cell character while simultaneously allowing them to generate progeny that undergo amplification and differentiation. Studies from both Drosophila germ cells and vertebrate hematopoetic and epithelial stem cells indicate that retaining stem cells in their niches requires interactions of stem cells and surrounding cells through adherens junctions and integrins (Watt and Hogan, 2000; Fuchs et al, 2004). In the Drosophila ovary, the adherens junctions proteins, DE-cadherin and ?-catenin, are concentrated at the border of the germ cells and cap cells. Mutations in these molecules result in a failure to recruit and maintain germ cells (Song et al, 2002). In the bone marrow, maintenance of HSCs relies on their adherence to osteoblasts of the stroma through N-cadherin of adherens junctions and fibronectin-binding integrins (?4?1, ?5?1) (Zhang et al, 2003). Blocking this adhesion inhibits hematopoiesis in long-term bone marrow cultures (Whetton and Graham, 1999). Down-regulation of integrins is associated with loss of adherence of epidermal stem cells to the basement membrane (Watt and Hogan, 2000).
Activation of stem cells to divide may take place differently in maintenance vs. injury-induced regeneration. In maintenance regeneration, the stem cells undergo continual but slow division in response to environmental signals, feeding a constant excess of progeny into a new environment that induces them to differentiate. In injured tissues that do not normally undergo rapid turnover, stem cells remain dormant until injury-induced signals mobilize them.
Whether or not an epithelial stem cell divides symmetrically or asymmetrically appears to depend on whether intercellular contacts between stem cells, or contacts between stem cells and basement membrane of the epithelium is stronger. In the former case, the mitotic spindle lines up perpendicular to the basement membrane, cell fate determinants are equally segregated into the halves of the cell, and two identical stem cells are produced. In the latter case, the spindle lines up parallel to the basement membrane, cell fate determinants are distributed unequally, and the result is a stem cell adherent to the basement membrane and a free lineage-committed daughter (Yamashita et al, 2003).
A current controversy is whether the developmental potential of some adult stem cells is greater than their prospective significance. That is, can they be reprogrammed to transdifferentiate by extracellular signals from other cells (Fuchs and Segre, 2000; Weissmann, 2000; Stocum, 2004a)? If so, it would be possible to use just one type of autogeneic stem cell, say from the easily accessible bone marrow, for transplantation to a lesion site to generate the cell types that make up the local tissue. Recent evidence, however, suggests that experimental results that have been interpreted as transdifferentiation are actually due to fusion with host cells (Alvarez-Dolado et al, 2003).
On the other hand, there is evidence that the bone marrow and connective tissue compartments of multiple organs harbor multipotent and/or pluripotent stem cells that share a number of qualities with ESCs, though they are different from ESCs (Jiang et al, 2002; Young and Black, 2004). These cells have been isolated after extensive culturing of bone marrow and connective tissue cells and have been shown to differentiate into many, if not all, cell types, both in vivo and in vitro. Whether these cells actually exist as such in the in the bone marrow or connective tissue or are the result of dedifferentiation in culture is not yet clear. Either way, their use in regenerative medicine would obviate two of the major problems of ESCs, the need to combat immunorejection and the bioethical issues associated with the production of ESCs.
Creation of stem cells via dedifferention
Dedifferentiation is a loss of phenotypic specialization that converts differentiated cells into adult stem cells, which then proliferate and differentiate into replacement tissue (Brockes, 1998; Stocum 2004b). Dedifferentiation is a relatively common mechanism of regeneration in lower vertebrates. Fish can regenerate fins and barbels (teleosts) by dedifferentiation (Geraudie et al, 1998), and certain species of lizards can regenerate tails by this mechanism. The divas of dedifferentiation in the vertebrate world, however, are the anuran tadpoles and the larval and adult urodele amphibians (Carlson, 1998). These animals can regenerate the same tissues as mammals via compensatory hyperplasia and reserve adult stem cells, but use dedifferentiation to regenerate a wide variety of tissues and complex structures that mammals cannot regenerate, including lens, neural retina, and intestine (Stocum, 1995). The amphibians are also exceptional in their ability to regenerate complex structures such as limbs, tails, and jaws (Brockes, 1997; Carlson, 1998; Stocum 2004b). Mammals are unable to regenerate amputated tails or appendages, but there are exceptions :- deer antlers, the distal phalanges of mice and humans, holes in rabbit ears, and holes in bat wings (Stocum, 1995).
The regeneration of complex structures such as appendages is an epimorphic process. Morgan (1901) distinguished two modes of regeneration, morphallaxis and epimorphosis, that are independent of the types of regeneration-competent cells used for regeneration. Morphallaxis is the regeneration of missing parts in the absence of growth, by repatterning the remaining tissue into a normally proportioned but smaller whole, followed by growth to the original size. It is typical of regeneration in multicellular animals with relatively simple tissue organizations, such as Hydra. Epimorphic regeneration restores body parts by the proliferation of stem or progenitor cells, followed by their differentiation into the tissues that were lost. Differentiation is thus linked to growth in epimorphic regeneration, whereas it is not in morphallactic regeneration. In addition to limbs and antlers, epimorphic regeneration is characteristic of flatworms and annelid worms (Goss, 1969).
Research Strategies and Experimental Models in Regenerative Biology
The ultimate objective of regenerative biology is to define the permissive and inhibitory factors that determine whether regeneration or fibrosis will take place after injury. This knowledge can then be used to devise pharmaceutical therapies to stimulate the regeneration of damaged human tissues that do not regenerate spontaneously, or whose regenerative capacity has been compromised. Currently, we know little about the differences that lead to regeneration as opposed to fibrosis. The failure of a tissue to regenerate could be due either to a lack of regeneration-competent cells, the lack of an environment favorable to regeneration, or both. The fact that regeneration-competent cells can exist as both differentiated or undifferentiated reserve cells suggests that virtually all adult cells have the potential to engage in regeneration, an idea that is compatible with the presence in all cells, with the exception of B and T cells, of a complete genome. This genome can, under natural and experimental circumstances, be reprogrammed to regenerate tissues and appendages (Stocum, 2004b) or even complete organisms (Byrne et al, 2002; Wilmut et al, 2002).
The standard approach to understanding why regeneration fails in mammalian tissues that do not regenerate spontaneously is to lesion these tissues, attempt to identify molecules in the injury environment that are inhibitory to regeneration, and devise ways to neutralize them. While this approach is useful, a more direct strategy is to compare and contrast the transcriptomes and proteomes of regenerating versus non-regenerating tissues and define the stimulatory and inhibitory molecules that differentiate regeneration from fibrosis. There are three experimental models that can be used for these comparisons. The first model compares wild-type tissues to genetic variations that confer a gain or loss of regenerative capacity. For example, there are several strains of mice that can regenerate ear and heart tissue (Heber-Katz et al, 2004), whereas the short-toes mutant of the axolotl causes deficiencies in limb regeneration (Humphrey, 1967). The second model is to compare the same tissues at developmental stages when they are capable of regeneration versus stages when they are not. For example, fetal skin in many mammalian species regenerates perfectly, but late in gestation the injury response switches to scar tissue formation characteristic of the adult (McCallion and Ferguson, 1996). The third model compares the same tissue between two species, one of which regenerates the tissue and the other does not. For example, the ability of cultured myotubes to re-enter the cell cycle in response to serum factors has been compared in the newt and mouse (Tanaka et al, 1999).
The amphibians are particularly useful for the latter two comparative strategies. Frogs regenerate many tissues well prior to metamorphosis, but lose the capacity to regenerate at late tadpole stages or by the time metamorphosis is complete (Stocum, 1995). Thus, cellular and molecular comparisons can be made between tissues at regeneration-competent and regeneration-deficient stages of development. Or, regeneration-competent juvenile or adult urodele tissues can be compared with regeneration-deficient anuran tissues. Genomic studies have repeatedly shown that developmentally important genes are strongly conserved across vast evolutionary distances. Much of what we know of vertebrate developmental and molecular genetics has been derived from studies on Drosophila and other invertebrates. Thus it is not unreasonable to expect that the same genes involved in the regeneration of amphibians are conserved in humans, though their activity is either suppressed or neutralized by inhibitory factors.
Evolutionary Significance of Regeneration and Fibrosis
Wound repair and tissue regeneration are ubiquitous to multicellular organisms. These processes are universally adaptive, in that they are obligatory for the survival of multicellular organisms; thus natural selection will always favor them. Epimorphic regeneration, however, is restricted to only a relatively few species within each phylum. This fact is sometimes taken to mean that epimorphic regeneration arose in a few species from non-regenerating ancestors by selection for favorable mutations conferring this ability. However, Goss (1987; 1992) has pointed out that the de novo origin of epimorphic regeneration by positive selection is improbable, because it would have had to be an all or nothing event involving at least several simultaneous mutations.
Regeneration can be viewed as a default state of stem cells that is suppressed in many tissues of adults. This state is similar to the regulative ability of undifferentiated cells of the embryo to compensate for the loss, addition, or scrambling of cells. This notion is consistent with the fact that many non-regenerating tissues harbor stem cells (see below), with the similarity of regenerative mechanisms in the organisms of different phyla, and with the fact that regeneration appears in most cases to recapitulate embryological processes. For example, the growth, differentiation and patterning of the urodele limb regeneration blastema is superficially indistinguishable from the development of the urodele embryonic limb bud.
Why has regeneration been suppressed in favor of fibrosis in the adult tissues and appendages of so many species? One argument is that it confers no adaptive advantage, even in those species that possess it (Goss, 1987; 1992). Regeneration may require considerably more energy expenditure over a longer period of time than fibrosis, particularly with regard to large and complex structures such as limbs. In mammals, which are warm-blooded and whose injured tissues are particularly susceptible to bacterial infection and water loss, rapid wound closure and scar formation would be particularly advantageous because it prevents fluid loss, suppresses bacterial proliferation and provides a relatively quick patch to the wound.
The most logical conclusion, therefore, is that regeneration is suppressed in most adults because they exist under conditions where fibrosis is more energy efficient and provides more assurance of survival. This suppression has been related to the maturity of the immune system in both fetal vs. adult mammalian skin and limb regeneration in the frog, Xenopus (McCallion and Ferguson, 1996; Harty et al, 2003).
Regenerative Medicine
Currently, the only way we have to compensate for diseased or injured tissues is through bionic implants and organ transplants. Attempts to establish a regenerative medicine have been ongoing for over two decades. The vast majority of work has been done with experimental animals, although human clinical trials to restore several types of tissues, such as islet cells of the pancreas to cure diabetes and dopaminergic neurons of the substantia nigra to cure Parkinson’s disease, have been made as well. Three strategies have been used: cell transplants, the construction in vitro of implantable bioartificial tissues (“tissue engineering”) and the induction of regeneration in vivo. These strategies are diagrammed in Figures 1 and 2.
Cell Transplants
This approach involves replacing the cells of a damaged tissue by transplanting aggregates of donor cells into the lesion. The transplanted cells can be differentiated cells, embryonic stem cell derivatives, or adult stem cells. The cells can be normal or genetically modified to boost production of physiologically important molecules, or molecules designed to neutralize factors inhibitory to cell survival and proliferation.
Differentiated cells can be harvested directly from donors, but with few exceptions, their expansion in vitro is difficult. Alternatively, differentiated cells or their precursors could be generated in vitro from human ESCs, or from ASCs. A major advantage of ESCs is that they can be expanded indefinitely in culture without losing their potential for differentiation. Cells harvested from donors or differentiated from ESCs, however, are allogeneic and therefore subject to immune rejection, unless transplanted to an immunoprivileged site. Another problem is that we cannot, at present, precisely direct the differentiation of ESCs into most cell types. Furthermore, their use (as well as that of fetal cells) is surrounded by bioethical and legal controversy, because their production requires the destruction of a human embryo. Adult stem cells can be harvested from the patient and therefore have the advantage of not provoking an immune reaction or generating ethical controversy. To use specific ASCs, however, we need to know how easy they are to expand in vitro, what factors are necessary for their differentiation and tissue organization, and whether these factors are present in the injury environment of the tissue we wish to regenerate.
Cell transplantation has been modestly successful in experimental animals and humans in treating neurological disorders such as Parkinson’s disease (Bjorklund and Lindvall, 2000; Lindvall and Hagell, 2001; Kim et al, 2002; Ourednik et al, 2002; Marconi et al, 2003) , multiple sclerosis (Pluchino et al, 2003), spinal cord injury (Teng et al, 2003), diabetes (Shapiro et al, 2000), myocardial infarction (Menasche, 2002; Beltrami et al, 2003), and damage to articular cartilage (Brittberg et al, 1994). In cases where adult neural stem cells have been used to attempt axon regeneration after spinal cord injuries or in Parkinson’s disease, there is strong evidence that the implanted cells themselves provide only a modest degree of regeneration. The majority of the regenerated axons or new neurons were from host cells, indicating that the donor stem cells produce survival and outgrowth-promoting factors that protect host cells and enable them to engage in regeneration (Kordower et al, 2000; Ourednik et al, 2002; Marconi et al, 2003).
Bioartificial Tissues
The concept of bio-artificial tissue construction (tissue engineering) is to seed cells into a biomaterial scaffold, then implant this construct into the body (Nerem and Sambanis, 1995). The constructs can be open (scaffold molded in the shape of the tissue, open to vascularization by the host) or closed (cells encapsulated and dependent on diffusion for survival). Everything that applies to what has been said about cells used for transplantation applies to the cells used in the construction of bioartificial tissues as well. Ideally, scaffolds would be highly biomimetic, not only providing the proper geometry and adhesive qualities to maximize cell migration, but also incorporating the biological cues and signals essential for proliferation and differentiation, as well as any factors for neutralization of molecules inhibitory to regeneration. Scaffolds should also be biodegradable on a schedule that matches the growth and differentiation of the regenerating tissue. Widely used biomaterial scaffolds are collagen I, alginate, ceramics, polylactic and polyglycolic acid meshes, and pig small intestine submucosa (SIS) (Badylak, 2002; Langer and Tirell, 2004). No existing biomaterial meets all the criteria deemed necessary for good tissue regeneration, however, and research on new materials is of high priority.
Closed constructs require the use of differentiated cells, whereas open constructs can use precursors derived from ESCs or use ASCs. If the cells of an open construct are allogeneic, they will be subject to immunorejection; those of a closed construct will not. Other challenges to building open constructs include incorporation of angiogenic factors, as well as proliferation and differentiation factors, into the scaffold and creating three-dimensional patterns of cells that allow for maximum diffusion while blood vessels grow into the proliferating tissue.
Several types of bioartificial tissues have been tested for replacement of tissues in experimental animals and humans. Biortificial skin equivalents are in wide use to treat burns, large excisional wounds, and venous and diabetic ulcers, bioartificial bone has been used to bridge large surgically created gaps in bone, and success has been had in fashioning digestive and genitourinary constructs (Nerem and Sambanis, 1995; Atala, 1997; Ogle et al, 1998).
Chemical Induction of Regeneration In Vivo
This strategy involves the use of combinations of regeneration-permissive molecules and neutralizers of regeneration-inhibiting molecules to stimulate regeneration from the body’s own tissues (Stocum, 2004a). The idea is to activate and recruit local regeneration-competent ASCs that would form scar tissue in the absence of intervention, or induce regeneration-incompetent cells to become competent, perhaps by stimulating compensatory hyperplasia or the dedifferentiation of mature cells. The regeneration-permissive molecules might be delivered as soluble “molecular cocktails” or as part of a cell-free regeneration template (scaffold), into which local or circulating cells would migrate. The advantage of this approach is that it eliminates problems of donor availability, immunorejection, and bioethical concerns in one stroke, and would be relatively low-cost.
There is substantial evidence that many mammalian tissues house ASCs that normally are not activated or participate in scar tissue formation after injury, indicating that mammals have considerable latent capacity for regeneration that is suppressed (Stocum, 2004a). Furthermore, both the spinal cord and the heart initiate regeneration that is then aborted by inhibitory factors in the injury environment and the formation of scar tissue. Thus, by changing a non-permissive injury environment to a regeneration-permissive one, we may be able to initiate and/or complete the regenerative process.
A number of non-regenerating tissues have been induced to undergo some regeneration in experimental animals. Biodegradable, cell-free artificial regeneration templates have been used to induce dermal regeneration in excisional skin wounds and regeneration of peripheral nerves, though the regeneration is imperfect (Yannas, 2001). A variety of neuroprotective agents, agents that neutralize molecules inhibitory to axon regeneration, and enzymes that degrade glial scar, have been used to treat spinal cord injury and Parkinson’s disease (Stocum, 2004a). Cell-free ceramic templates can induce bone regeneration across gaps (Constanz et al, 1995; Yaszemski et al, 1995). Attempts to induce limb regeneration from the non-regenerating limbs of adult frogs or the digits of mice have resulted only in a slight regenerative response (Stocum, 1995).
The induction of regeneration from the body’s own tissues, like cell transplantation and bioartificial tissue implants, requires that we understand what signals are required to activate and promote proliferation, differentiation, and tissue organization of regeneration-competent cells, to determine whether or not these signals are present in the injury environment, and to identify what inhibitory factors might be present in the injury environment that need to be suppressed or neutralized. This understanding is currently being gained by experiments comparing the cellular and molecular differences between regeneration and scarring in experimental systems such as fetal vs. adult skin wound repair in mammals (Gosiewska et al, 2001), and limb regeneration in early vs late frog tadpoles (King et al, 2003).
Regenerative Biology and Medicine: A Millennial Revolution
Bionics and organ transplantation will advance and continue to be important ways of restoring structure and function in the 21st century, but regenerative biology and medicine promises to be one of the biomedical revolutions of the millennium. The goal is to be able to replace organs and appendages with bioartificial constructs or to guide the repair process along a regenerative pathway, rather than a pathway leading to scar tissue formation. The advancing cellular and molecular knowledge of the differences between regeneration and fibrosis, the fact that mammals have hidden regenerative capacity that is suppressed, and the success in treating some conditions by cell therapy, bioartificial tissues, and molecular agents, has strengthened our confidence that within a decade or two, we will be able to add regeneration to our repertoire of medical treatments.
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