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Can Vet J. 2008 October; 49(10): 1009–1017. | PMCID: PMC2553494 |
Copyright and/or publishing rights held by the Canadian Veterinary Medical Association Concepts for the clinical use of stem cells in equine medicine Thomas G. Koch, Lise C. Berg, and Dean H. Betts Department of Biomedical Sciences, University of Guelph, Guelph, Ontario N1G 2W1 (Koch, Betts); Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Grønnegårdsvej 7, 1870 Frederiksberg C, Denmark (Koch, Berg) Stem cells from various tissues hold great promise for their therapeutic use in horses, but so far efficacy or proof-of-principle has not been established. The basic characteristics and properties of various equine stem cells remain largely unknown, despite their increasingly widespread experimental and empirical commercial use. A better understanding of equine stem cell biology and concepts is needed in order to develop and evaluate rational clinical applications in the horse. Controlled, well-designed studies of the basic biologic characteristics and properties of these cells are needed to move this new equine research field forward. Stem cell research in the horse has exciting equine specific and comparative perspectives that will most likely benefit the health of horses and, potentially, humans. Concepts pour l’utilisation clinique des cellules souches en médecine équine. Les cellules souches provenant de différents tissus suscitent de grands espoirs en thérapeutique équine mais jusqu’à maintenant la preuve de leur efficacité ou le principe même de leur utilité n’a pas été établi. Les caractéristiques de base et les propriétés des diverses cellules souches équines demeurent largement inconnues en dépit d’une augmentation des indications expérimentales et d’une utilisation commerciale empirique. Une meilleure compréhension de la biologie des cellules souches équines et des concepts impliqués est nécessaire pour développer et évaluer des applications cliniques rationnelles chez le cheval. Des études contrôlées et bien planifiées sur les caractéristiques et les propriétés biologiques de base de ces cellules sont nécessaires pour faire évoluer ce nouveau champ de recherche. La recherche sur les cellules souches du cheval ouvre des perspectives enlevantes tant pour l’espèce équine que pour la médecine comparative. Ces avancées pourront vraisemblablement améliorer la santé équine et, potentiellement, la santé humaine. (Traduit par Docteur André Blouin) Stem cells, especially human embryonic stem cells, have sparked huge public and scientific interest, given that these cells are known to have unlimited potential for growth and differentiation into most, if not all, cell types of the adult body ( 1– 3). Recently, embryonic stem-like cells have been derived from equine blastocysts produced in vivo and then differentiated in vitro ( 4– 7). Stem cells from bone marrow, fat tissue, the umbilical cord, and umbilical cord blood are currently being used experimentally and commercially in the horse ( 8– 11). Proof-of-principle that cell based treatment regimes and their professed empirical efficacy are, in fact, caused by stem cells, and not by other cells and/or biological factors administered simultaneously, has not been proven. The efficacy and safety of these treatments and the basic characteristics of the stem cells used remain largely unknown. The purpose of this review is to provide basic concepts of stem cells and the potential therapeutic and adverse effects of stem cell-based therapies. Where available, knowledge gained in the horse will be discussed. Medline, the Commonwealth Animal Bureaux (CAB), Agricola PlusText, and the World Wide Web search engine Google were used to collect and review most of the references. The keywords used in the search of the databases were stem cells, mesenchymal, umbilical cord blood, umbilical cord matrix, Wharton’s jelly, immunogenicity, cell tracking, cryopreservation, safety, equine, lameness, arthritis, osteoarthritis, tendon, tendonitis, tissue engineering, biological scaffolds, embryonic stem cells, stem cell stemness, stem cell plasticity, stem cell function, and transdifferentiation. In addition, papers for review were identified from the reference lists of other papers, or through personal knowledge of reports or conference proceedings. Only peer-reviewed articles have been included in the reference list. Stem cell definition, “stemness,” and stem cell nomenclature Stem cells are defined as having the in vivo capacity to self-renew, to proliferate extensively, if not indefinitely, and to differentiate into 1 or more cell/tissue types () ( 1, 12, 13). In theory, these cells should adhere to Koch’s postulates for disease-causing organisms, in the sense that they can be implanted and then recovered from the tissue after a period of time, when they will show the same degree of differentiating ability. The ability to differentiate is known as “potency.” Potency is classified as uni-, multi-, pluri-, and totipotency, depending on the number of different tissue types the stem cell can produce. Unipotent stem cells are generally the residing stem cells of certain adult tissue within a specific organ (skin, liver, intestine) that are responsible for the ongoing renewal of these tissues. Multipotent stem cells can give rise to multiple differentiated cells or tissues. A good example is the mesenchymal stem cell (MSC), which contains classic trilineage differentiation potential to chondrogenic, adipogenic, and osteogenic lineages. These 3 cell fates all originate from the embryonic mesoderm. Multipotency is used less frequently to describe stem cells capable of giving rise to cell lineages of 2 different germ layers, since there is no defined terminology for cells capable of differentiating into only endoderm (hepatocytes and other gastrointestinal tissues) and mesoderm (musculoskeletal tissues) cell lineages, endoderm and ectoderm (nervous tissue) cell lineages, or mesoderm and ectoderm cell lineages. Embryonic stem cells are regarded as pluripotent, since they can give rise to all the 3 germ layers, including cells of the germline, in principle the whole organism. A totipotent stem cell is capable of generating any cell in the body, including those of the extra-embryonic tissues and the fetal tissues of the placental unit. So far, the zygote and cells from the very early embryonic stages are the only known totipotent stem cells; the cells from the inner cell mass (ICM) of the blastocyst are regarded as being pluripotent, not totipotent ( 14). However, human embryonic stem cells have been shown to give rise to extra-embryonic trophoblasts, suggesting that embryonic stem cells may be able to give rise to some placental cell types ( 15, 16). Further studies are needed to validate these results and rule out the possibility of cellular “contamination” of the ICM with cells from the trophectoderm in the initial establishment of the embryonic stem cell lines. Despite these relatively straightforward definitions of stem cells and potency, controversy often arises when trying to prove stemness and potency of specific cell types. The characteristics of stemness, as outlined previously, can be difficult to prove due to the lack of specific molecular or genetic markers of stem cell properties; therefore, the definition of stemness is currently, to a large degree, hypothetical ( 17, 18). Until stringent objective parameters characteristic of stem cells are established, this debate will likely continue. In the meantime, proliferative capacity and differentiation ability (in vitro and/or in vivo) will largely determine whether a stem cell-like claim can be made. Besides temporal categorization into embryonic, fetal, or adult, stem cells are often further grouped, based on tissue origin, potency, and possible cell surface markers. In the past 2 decades, adult stem cells have often been grouped into hematopoietic or mesenchymal stem cells (MSC), based on their differential potential. Mesenchymal stem cells are sometimes referred to as stromal stem cells or stromal bone marrow stem cells, since they were first isolated and described from bone marrow aspirates ( 19). Known surface markers have been identified for hematopoietic stem cells that are cluster of differentiation (CD)14, CD34, and CD45 positive ( 20). This knowledge can be utilized to quickly separate these stem cells from a mixed cell suspension by using automated fluorescence-activated cell-sorting (FACS) techniques ( 21). The International Society for Cellular Therapy has recently reviewed the general term “mesenchymal stem cells” and has introduced the name “multipotent mesenchymal stromal cells” ( 22, 23). Human multipotent mesenchymal stromal cells are cells that adhere to plastic, express the surface markers CD73, CD90, and CD105, do not express CD45, CD34, CD14 [CD11b], CD79 [CD19], and HLA-DR, and have trilineage differentiation potential towards osteoblasts, adipocytes, and chondroblasts. The term mesenchymal stem cell should be reserved for cells with in vivo demonstrations of long-term survival with self-renewal capacity and tissue repopulation with multilineage differentiation ( 23). The purpose of redefining the mesenchymal nomenclature is 2-fold: first, a precise description of cell populations will allow for a more valid comparison of research results from different investigators; second, the term “stem” in mesenchymal stem cells might infer more biological and functional properties than the cells actually possess, which might lead to unrealistic expectation for these cells, especially in the lay literature. The acronym MSC is now nonspecific and is used for both cell populations. When reporting future MSC studies, investigators are encouraged to clarify which of the 2 cell types their work involves. The distinction between these 2 stem cell populations has not been universally accepted; therefore, in this review, the use of the term mesenchymal stem cells, as reported by the investigators, has been maintained, although many of the reports are, in fact, more correctly reporting multi-potent mesenchymal stromal cells, according to the International Society for Cellular Therapy. Nonhuman cells might not have the same surface marker profile as human MSCs, so, for now, adherence to plastic and trilineage potential is likely sufficient to identify multipotent mesenchymal stromal cells in the horse and other domestic species. Stem cell sources, isolation, and expansion Stem cells are often categorized into embryonic, fetal, or adult in origin, based on the age of the donor tissue; overlap between these categories exists. Is a stem cell that is derived from the umbilical cord tissue or umbilical cord blood a fetal, an adult, or, maybe more precisely, a neonatal stem cell? Embryonic stem cells most commonly originate from the ICM of the blastocyst () ( 1, 12). The ICM is isolated by mechanical dissection; immunodissection, where trophectoderm specific antibodies, in combination with serum-derived complement factors, cause lysis of the trophectoderm; or a combination of both. The ICM is then cultured in tissue dishes containing a mitotically inactivated feeder cell layer, which, generally, is of murine fetal fibroblast origin. Feeder-layer cells have been essential for the isolation of embryonic stem cells by largely unknown mechanisms. Likely, the feeder-layer secretes growth factors and mediators needed by the embryonic stem cells. Expansion and, in a few reports, derivation of human embryonic stem cells can now be done without a feeder-layer and without the use of animal sera ( 24– 26). This practice avoids possible xenobiological contamination, which is an immunological transplantation safety concern in the clinical application of embryonic stem cells. The initial blastocyst can be produced in vivo after natural or artificial insemination, in vitro by in vitro fertilization (IVF), by direct sperm injection, or even by somatic cell nuclear transfer (cloning). Human embryonic stem cell lines have also been derived from single blastomeres ( 27). These studies demonstrate the possibility of creating patient specific stem cells with fewer ethical concerns and restrictions than are raised by human therapeutic cloning and the “destroying” of human embryos. Most recently, mouse skin fibroblasts have been ‘induced’ to become pluripotent stem cells with similar potency as embryonic stem cells ( 28– 30). These cells were produced by transfecting skin fibroblast with the genes coding for Oct 3/4, Sox 2, c-Myc, Klf4 under embryonic stem cell culture conditions and have been named induced pluripotent stem (iPS) cells. Fetal, adult, and neonatal stem cells are isolated by separating out the mononuclear cell fraction (MNCF) from the tissue of interest () ( 11, 13, 21, 31, 32). To do this, mechanical and enzymatic digestion, followed by centrifugation using density medium, may be required. In contrast to embryonic stem cells, these stem cells do not require a cell feeder layer and can be plated directly onto plastic culture dishes or flasks. This approach takes advantage of the propensity of these stem cells to adhere to plastic surfaces, such as polystyrene. With the appropriate culture medium, colonies of adherent cells are noted within days to weeks. As the cells approach confluency, they can be detached enzymatically from the surface of the culture dish and brought into suspension. The cell suspension can then be divided between multiple culture flasks for expansion of the number of undifferentiated cells or for in vitro differentiation towards specific cell lineages for therapeutic use. In some instances, the isolated mononuclear cell fraction is used therapeutically without isolation and expansion of the stem cell numbers. “Stromal-vascular fraction” (SVF) is the term often used to describe the nucleated cell fraction isolated from adipose tissue ( 33– 35). Stem cell plasticity and transdifferentiation The possibility that adult tissue-specific (unipotent) stem cells could change phenotype leads to the debate regarding stem cell plasticity or transdifferentiation (). This concept remains controversial, since it conflicts with the belief that when a stem cell population begins to differentiate towards a certain cell lineage, there is no return; that is, the stem cell progeny cells are committed to a specific cell lineage and have become unipotent ( 36– 39). The apparent change in phenotype, based on expression of new surface markers, may be due to fusion between a stem cell and a somatic cell, which would create a cell with different surface markers and phenotype ( 37, 39). The ability of somatic cells to fuse is well known and is the basis for the commercial production of monoclonal antibodies from hybridomas ( 40). Actual plasticity or transdifferentiation could be due to several mechanisms, including the dedifferentiation of a differentiated cell into a multipotent stem cell, which subsequently undergoes differentiation towards 1 or more new cell lineages ( 41). Homing, engraftment, and niche Homing describes the observation that some stem cells seed in preferred tissues after intravenous injection ( 42– 44). The translocation of immunoglobulin A producing plasma cells from the gut-associated lymphoid tissue to the mammary glands of pregnant animals is an example of normal physiologic homing. Engraftment describes the ability of stem cells to invade and be incorporated into tissues and organs. Notably, engraftment does not necessarily imply function. The term stem cell “niche” refers to the local environment of the stem cells, which has crucial regulatory functions due to largely unknown factors, likely acting in autocrine and paracrine fashions to maintain stemness and promote differentiation. Treatment of children suffering from various hereditary diseases relies on these 3 concepts. Allogenic hematopoietic stem cells from either bone marrow or umbilical cord blood are injected intravenously into the child to restore normal myeloid function, erythroid function, or both ( 45). The injected stem cells home to the bone marrow and engraft, the niche then guides the stem cells towards hematopoietic cell lineages. Immune rejection, especially in graft versus host disease, is a valid concern when allogenic cells (from a different individual) rather than autologous cells (from the same individual) are used. However, the use of autologous stem cells might not always be feasible, due to donor site morbidity, or desirable, in cases of hereditary diseases ( 19). In addition, autologous stem cells from aged individuals may not have the same proliferative and differential capacity as fetal, neonatal, and embryonic stem cells ( 46). Allogenic, hematopoietic bone marrow-derived stem cells generally need to be tissue-matched to the recipient and they carry the risks of any allogenic grafts of transmitting infectious and noninfectious disease ( 45). Nontissue-matched, allogenic, cord blood-derived hematopoietic stem cells have been administered to children without apparent immunological reactions ( 45). Whether the apparent immune tolerance in these cases is due to a somehow immature immune system in young patients or to cellular characteristics of the cord blood-derived stem cells is debatable. The immunogenicity of adult human mesenchymal stem cells derived from bone marrow is incompletely understood, but these stem cells appear to be hypo-immunogenic and to exert significant suppression of T-cell and dendritic cell functions in vitro and in vivo, leading to evasion of allorejection and, in some instances, systemic immune modulation ( 47– 49). In-depth discussion of bone marrow-derived mesenchymal stem cells is beyond the scope of this review, but is available elsewhere ( 20, 47, 48, 50). In a baboon model of tissue mismatched baboon skin grafts, the intravenous administration of baboon bone marrow-derived mesenchymal stem cells from the same baboon as the skin graft was harvested from (the donor animal of BM-MSCs and skin grafts) caused delayed host rejection by the recipient baboon of the allogenic skin grafts, indicating an immune modulating effect of the infused donor BM-MSCs ( 51). Patients suffering from therapy-resistant graft-versus-host disease have been treated successfully with allogenic bone marrow-derived mesenchymal stem cells ( 52). Based on these encouraging results, it is now speculated that bone marrow-derived mesenchymal stem cells may be used as immunotherapy for inflammatory bowel disease and other inflammatory diseases ( 53, 54). However, both B-cell stimulation and suppression have been noted in vitro after exposure to bone marrow-derived mesenchymal stem cells ( 55, 56). It remains to be shown conclusively whether the noted in vivo immune-modulatory effects are related solely to the patient population studied (children versus adults, immune-competent versus immune-compromised, or healthy versus diseased), the stem cell characteristics, or a combination of both. It is also unknown, at present, if the apparent positive effects on the immune system persist long-term and whether serious adverse immunologic associated effects will occur later. Today, embryonic stem cells are allogenic in nature due to the destruction of the blastocyst in the process of isolating the embryonic stem cells. The general perception is that allogenic embryonic stem cells will be rejected if transplanted into an immune competent host, although recent studies of human embryonic stem cells transplanted into immune-competent mice have indicated that embryonic stem cells may become immune-privileged under certain conditions ( 57). Currently, 2 technologies might circumvent the problem of immune rejection altogether by creating autologous embryonic stem cells. The more established technique is somatic cell nuclear transfer. Isolation of embryonic stem cells from a cloned blastocyst reconstructed from a patient’s somatic cell would be regarded as nearly autologous in nature, thereby reducing the risk of immune rejection. Mitochondrial DNA in the enucleated oocyte and unknown epigenetic factors are the reasons why the cloned cells will not be an exact copy of the donor individual and this could possibly cause immune reactions. However, skin grafts between identical cloned pigs were not rejected, indicating tissue match ( 58– 60). The other option involves taking a biopsy of 1 or several cells from the ICM of a blastocyst without destroying the blastocyst ( 27). In theory, embryonic stem cells may then be established from the biopsied cells and the blastocyst could be transferred to the uterus of a recipient female. The resulting individual would at the time of birth have complete autologous embryonic stem cell line(s) available. This sequence of events is unlikely to occur in human medicine, but it may be an option in equine medicine. A third option to overcome immune rejection may be available with the use of iPS cells (see previous text), which hold enormous potential for advancing our knowledge of basic stem cell biology and, long-term, may offer a very potent autologous stem cell source for clinical use ( 29, 30). In vitro stem cell differentiation Stem cells can be coaxed to differentiate into various cell types in vitro by adding known growth and differentiation factors to the medium () ( 11). Most in vitro differentiation studies are performed on cells grown in a monolayer, but for some tissue types, such as cartilage, culture in 3-dimensional systems or exposing the cells to mechanical stimulation by cyclic pressure or ultrasound is required to enhance differentiation ( 13, 61– 63). In vitro differentiation studies are obviously useful to evaluate the differential potential of stem cells for basic characterization purposes and to determine whether the stem cell in question has potential for tissue repair of a specific tissue type. For treatment purposes, it might be advantageous to differentiate the stem cell in vitro towards the injured tissue type prior to transplantation, as discussed in more detail later. The basic work of determining which growth and differentiation factors control different differentiation pathways may also offer valuable in vivo applications to recruit and control resident stem cells of the tissues without direct stem cell administration. Diagnostic use of stem cell populations The application of differentiated stem cells for individual (patient-specific) diagnostic, prognostic, and pharmacological purposes has been advocated and has very interesting prospectives ( 64). The notion is that the individual genome determines the secretion of biological factors, surface markers, and tissue integrative properties of stem cells. All these factors together determine the overall homeostasis of the cell and have been referred to as the “biological set point” ( 64). Caplan and Bruder ( 64) speculate that molecular or genetic markers in mesenchymal stem cells will allow early prediction of who will suffer later in life from mesenchymal tissue dysfunctions, such as osteoporosis and osteoarthritis. Intervention to maintain normal mesenchymal cell function could then be initiated before disease develops. An example of pharmacological use could be to isolate mesenchymal stem cells from a patient suffering from hepatitis and differentiate the stem cells into hepatocytes in vitro. Then drugs (antibiotics, anti-inflammatories, etc.) could be tested in vitro for their pharmacological effects on tissue with that particular patient’s biological set point. This might optimize drug efficacy and reduce the risk of adverse effects by enabling the development of individual dosing regimes to replace the current use of general dosing regimes. The defined prerequisite for “true” human-derived embryonic stem cells is their capacity to differentiate into teratomas upon injection into immune-compromised mice ( 1, 14). Supposedly, equine embryonic stem cells did not show teratoma formation capability ( 4– 6). Teratoma formation has only been described for human- and murine-derived embryonic stem cells and it is undetermined whether there are significant basic differences between murine and human embryonic stem cells and those from other mammals, or if the cell lines reported for the horse were established from cells of trophectoderm or endoderm origin and not from the epiblast of the ICM. Whether nonembryonic stem cells have teratoma forming capacity is unknown, but so far teratoma formation has not been observed at the injection sites of MSCs ( 65). Injection of human embryonic stem cells, human umbilical cord blood-derived mesenchymal-like stem cells, and human bone marrow-derived mesenchymal stem cells into fetal sheep before day 65 of gestation has been performed ( 21, 66– 69). The resulting lambs were shown to be chimeras with tissue integration of cells of human origin. Notably, function of these human-derived cells was also demonstrated, since albumin of human as well as ovine origin was detected in the lambs’ circulating blood ( 21). Human-derived Purkinje fibers were also found in the hearts of these lambs ( 21). Teratoma formation and the fetal sheep model are regarded as noninjury in vivo models and give important information on how stem cells may behave in physiologically normal individuals. Injury models, where an injury occurs spontaneously or is deliberately induced, are used to study stem cell behavior with respect to their repair or regenerative potential as therapeutic agents. Cartilage and tendon lesions are currently the equine injury models most used to evaluate proposed stem cell therapies ( 8, 70– 77). Proposed in vivo functions of mesenchymal stem cells at injury sites Studies of human MSCs suggest that the stem cells exert a significant trophic and immunosuppressive function by unknown secretory factors and that this mechanism is more important than functional tissue integration of the stem cells in tissue regeneration () ( 47). If the stem cells mainly have a governing and regulatory role, knowledge of the molecules they secrete may, in the future, pave the way for more conventional drug approaches of injectable differentiation and repair factors, making the administration of the stem cells themselves obsolete. Coadministration is another potential use of stem cells. In addition to the example of the allogenic skin grafts and BM-MSCs in baboons, coadministration of mesenchymal stem cells and hematopoietic stem cells has been shown to enhance engraftment of hematopoietic stem cells in a mouse model ( 78). Injection of human bone marrow-derived mononucleated cells containing both hematopoietic and mesenchymal progenitor cells appears to have a superior therapeutic effect in human patients with chronic coronary artery disease (myocardial infarct) and peripheral occlusive arterial disease ( 79, 80). A combination of coadministration of various progenitor cells, as well as secreted cytokines from the cells, is thought to cause the observed therapeutic effect. Controlled equine trials are needed to evaluate efficacy of current stem cell-based treatments and to answer some basic questions; for example, do the stem cells functionally incorporate into the repaired/regenerated tissue or do they excite a short-term governing role recruiting and controlling resident cells to repair/regenerate the tissue? If the administered stem cells do become integrated, is their effect(s) long-term? Tracking and identification of administered exogenous stem cells in the recipient Ways of tracking and identifying the administered cells, in vivo and at postmortem, are crucial tools required to answer some of the questions of basic stem cell biology and function. Noninvasive cell-labeling methods for in vivo “cell tracking” studies include direct labeling or transfection with a marker gene ( 81– 83). Transfection of stem cells with a gene coding for a product that can be monitored can be used to track cells. The gene and product expression can be either continuous or induced by exogenous drug administration (typically doxycycline). Marking of cells with green fluorescent protein (GFP) is an example of a classic research tool of cell tracking, and it can be a useful choice for equine stem cell studies. The main drawbacks of GFP traditionally have been that a tissue biopsy or euthanasia of the animal was required and that cell fusion phenomena might lead to false positive conclusions. If the administered stem cell were to fuse with a somatic cell, it might, incorrectly, appear as if the stem cell had differentiated and incorporated into the tissue. If the cell of interest also showed function, an incorrect conclusion of stem cell in vivo function could be drawn. The problem of cell fusion will apply to any cell tracking method, but newer methods, using GFP and labeled nanoparticles, allow in vivo tracking using different imaging modalities such as magnetic resonance imaging and optical or nuclear imaging ( 83). These technologies allow “cell traffic” monitoring in vivo in real-time, including tissue accumulation (homing and engraftment) and function (niche) ( 83). However, it is possible that these manipulations might affect the normal physiological behavior and function of the cells. Most studies using these new cell-tracking methods have been developed in small laboratory species; therefore, biological, technical, and financial issues might delay their use in equine stem cell studies. Long-term storage of stem cells Cryopreservation and long-term storage of embryonic stem cells has proven difficult and remains an unsolved problem, although new techniques show promising results ( 84, 85). The long-term cryotolerance of bone marrow-derived MSC is unknown. A human study of in vivo engraftment capacity in patients with hematopoietic disease showed no detrimental effect of bone marrow-derived hematopoietic stem cells after 2.0 to 7.8 y of cryopreservation ( 86). Human cord blood-derived hematopoietic stem cells showed normal in vitro functions after 5 y of cryopreservation ( 87). However, the frequency of isolation of mesenchymal-like stem cells from frozen mononuclear cell fractions of human umbilical cord blood was significantly lower than that from fresh nonfrozen mononuclear cell fractions; therefore, stem cell isolation and expansion is recommended prior to cryopreservation ( 88). The upper time limit for cryopreservation of stem cells is currently unknown. Safety considerations of stem cell-based therapies The safety, both short-term and particularly long-term, of stem cell technologies is largely unknown. Safety concerns to consider include, but are not restricted to, 1) aberrant cell development, and 2) tissue or vehicle contamination with infectious agents of foreign biological and nonbiological substances used in the laboratory during processing of the stem cells. The presence of neoplastic stem cells has been proposed as a source of neoplastic development, as well as the reason for the observed treatment failure in certain neoplasms ( 89). The rational is that though debulking, chemotherapy, and radiation therapy might kill and remove most neoplastic tissue, a few neoplastic cells with stem cell-like properties might escape these treatments. These neoplastic stem cells would then be able to form new tumors and cause clinical recurrence of disease. As mentioned previously, a requirement for authenticity of any embryonic stem cell is its capacity to form teratomas in immune-suppressed mice. The extent to which adult and neonatal stem cells can form such teratomas or other undesired tissue types is less well studied, but this possibility should be considered when and if stem cell-based therapies are being used ( 65). Transmission of infectious diseases is of special concern if allogenous cells are being used. Obviously, an animal with clinical or hematological signs of systemic disease is unlikely to be used as a stem cell donor for allogenic transplantation, but the possibility of adult stem cells harboring agents of latent chronic diseases has not been studied to the authors’ knowledge. The risks of infection associated with xenotransplant techniques have been studied. Studies evaluating the risk of viral transmission, in particular porcine endogenous retrovirus, from pig livers used for extracorporeal liver perfusion, showed that immune compromised mice did become infected, but immune-competent baboons did not ( 90, 91). The risk of infection from the use of allogenic stem cells relies on 2 main factors: the degree of epidemiological exposure of infections agents present in the allogenic material, and the net state of immune suppression of the patient ( 92, 93). 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