Recent advancements in developmental

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1 REVIEW A NEUROSURGEON S GUIDE TO STEM CELLS, CANCER STEM CELLS, AND BRAIN TUMOR STEM CELLS Samuel H. Cheshier, M.D., Ph.D. Stanford Institute of Stem Cell Biology and Regenerative Medicine, Departments of Neurosurgery and Developmental Biology, Stanford University School of Medicine, Stanford, California M. Yashar S. Kalani, M.D., Ph.D. Stanford Institute of Stem Cell Biology and Regenerative Medicine, Departments of Neurosurgery and Developmental Biology, Stanford University School of Medicine, Stanford, California STEM CELLS AND their potential applications have become the forefront of scientific, political, and ethical discourse. Whereas stem cells were long accepted as units of development and evolution, it is now becoming increasingly clear that they are also units of oncogenesis. Although the field of stem cell biology is expanding at an astounding rate, the data attained are not readily translatable for the physicians who may eventually deliver these tools to patients. Herein, we provide a brief review of stem cell and cancer stem cell biology and highlight the scientific and clinical implications of recent findings regarding the presence of cancer-forming stem cells in brain tumors. KEY WORDS: Brain tumor, Brain tumor stem cell, Cancer stem cell, Stem cell Neurosurgery 65: , 2009 DOI: /01.NEU A online.com Michael Lim, M.D. Department of Neurosurgery, The Johns Hopkins Hospital, Baltimore, Maryland Laurie Ailles, Ph.D. Stanford Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California Steven L. Huhn, M.D. Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, Stem Cells, Inc., Palo Alto, California Irving L. Weissman, M.D. Stanford Institute of Stem Cell Biology and Regenerative Medicine, Department of Developmental Biology, Stanford University School of Medicine, Stanford, California Reprint requests: Irving L. Weissman, M.D., 279 Campus Drive, B257 Beckman Center, Stanford, CA irv@stanford.edu Received, June 22, Accepted, April 1, Copyright 2009 by the Congress of Neurological Surgeons Recent advancements in developmental biology have brought stem cells into the forefront of scientific, political, and ethical discussions. Most clinicians have an intuitive sense of what stem cells are, but the significance of stem cell biology for their practice may not be fully appreciated. Recent experiments implicating stem cells as the source of cancers have led to new questions about the mechanisms of oncogenesis as well as new treatment strategies. We provide a broad overview of normal and cancer stem cell biology and highlight the scientific and clinical implications of recent findings regarding the presence of cancer-forming stem cells in brain tumors. STEM CELLS Understanding how a multicellular system develops from a single cell or cell type is one of the central issues of modern biology. The Greek philosopher Aristotle may have been the first to introduce the idea of stem cells when he debated whether the human embryo developed from a preformed individual (a homun - culus) or from an undifferentiated form that ABBREVIATIONS: AML, acute myelogenous leukemia; BM, bone marrow; BTSC, brain tumor stem cell; CNS, central nervous system; ES, embryonic stem; GBM, glioblastoma multiforme; HSC, hematopoietic stem cell; LT, long term; TS, tumorsphere gradually became more specialized into the many parts of a person. Although Aristotle eventually favored the homunculus, debate and controversy about human embryonic origins has continued into the modern era. It was not until 1665, when Robert Hooke observed the structure of cork bark under a microscope, that an accurate description of the cellular structure of an organism was obtained. From that time forward, cells were known to be the units of organization and function of tissues and organs. Because organisms live much longer than their differentiated cells, tissue and organ regeneration is necessary. We now know that most cells in a tissue or organ differentiate when they divide. Therefore, in a particular cellular lineage, the cells have a finite lifespan unless replaced. Stem cells replace these lost cells and are unique in that they self-renew themselves, providing a perpetual source of primitive precursors of the tissue or organ. Till and McCulloch (90) developed the formal concept of stem cells in the 1960s through a series of experiments involving the transplantation of limiting numbers of bone marrow (BM) cells, some of them chromosomally marked, into irradiated mice (90, ). At low numbers, 1 in 7000 marrow cells gave myeloerythroid (but not lymphoid) colonies 10 days later in the mouse spleen. Each colony was derived from a single cell, and some colonies produced more colony-forming cells; rarely, these included lymphoid progeny. We now call these blood colony-forming cells adult NEUROSURGERY VOLUME 65 NUMBER 2 AUGUST

2 CHESHIER ET AL. FIGURE 1. In the adult brain, stem cells reside in anatomic niches in the subventricular zone and the subgranular zone of the hippocampus. The neural stem cell is a cell capable of unlimited (depicted by the solid arrow) selfrenewal and differentiation to produce committed neural progenitor cells. Neural progenitor cells in turn are cells that also possess the ability to selfrenew, albeit to a more limited extent (depicted by the dashed arrow). Progenitor cells are capable of giving rise to lineage-specific progenitors that produce neurons, astrocytes, and oligodendrocytes. tissue stem cells, this type being hematopoietic. From these experiments, a general definition of stem cells emerged as cells possessing the following 3 characteristics: 1) self-renewal, 2) the ability to produce all cell types made in that tissue, and 3) the ability to do so for a significant portion of the life of the host (3). This definition has provided standard criteria to identify a stem cell. Figure 1 illustrates the definition of a stem cell as it pertains to the nervous system. The neural stem cell is a cell with an unlimited self-renewal potential. Through asymmetric cell division, the stem cell produces committed progeny or progenitors, which can also self-renew, but for a limited set of cell divisions, and differentiate to produce the various cell types of the central nervous system (CNS). Vertebrates contain numerous types of stem cells, some operative only at specific points during development, whereas others function throughout the lifetime of an organism. The most primitive, totipotent stem cells, are capable of giving rise to both the embryonic and extraembryonic tissues of an organism. Totipotent stem cells include the fertilized egg and the cells produced by the initial divisions of the ova. The product of these cell divisions, which in mammals occurs before the entity implants in the uterus, is the blastocyst. The blastocyst contains an outer sphere of trophoblast cells, capable of binding to and implanting into the uterus, and of helping form the placenta (Fig. 2). Within the blastocyst are 10 to 20 pluripotent cells called the inner cell mass. Upon implantation, these inner mass cells will participate in the production of all tissues and organs of the developing embryo, then fetus, then born organism. Such pluripotent cells can produce any differentiated cell in the body, but are usually unable to form the trophoblast cells. The best-known pluripotent stem cell is the embryonic stem (ES) cell. Although they are called ES cells, this is a misnomer; according to Dorland s Medical Dictionary, the embryo stage of development is well after the blastocyst stage: in humans, at about 2 weeks when the long axis forms a primitive streak until organogenesis at about 8 weeks when the fetal stage begins. In mice, introduction of ES cells into another mouse blastocyst allows chimerism of all tissues, including gametes; when such mice are mated, one can produce strains of mice from the ES donor. ES cells are obtained from the inner cell mass of the blastocyst and exist for only a brief stage of embryonic development. These ES cells can be manipulated in vitro, 238 VOLUME 65 NUMBER 2 AUGUST

3 A NEUROSURGEON S GUIDE TO STEM CELLS FIGURE 2. The union of a sperm and an egg produces the first true stem cell. This totipotent cell is capable of repeated divisions to produce the pluripotent blastocyst. Within the blastocyst reside some 20 cells, known as the inner cell mass, capable of giving rise to all the cells of the organism. Within the blastocyst, differential signaling environments allow for the formation of ectodermal, mesodermal, and endodermal stem cells that via committed differentiation steps produce tissue-specific stem cells such as those of the brain, skin, blood, muscle, gut, and the lung. in order to introduce new genes or disrupt preexisting genes within the ES cell genome. The widest application of ES cells has been to produce mice with targeted disruptions of specific genes known as knock-out mice. Given the ability of ES cells to produce any cell type of an adult organism, these cells offer great potential to be the instruments of research that can eventually lead to therapeutics in numerous human disease states. ES cell technology has already impacted scientific knowledge to the degree that its pioneer, Dr. Mario Capecchi, has been awarded a Nobel Prize. However, the fact that blastocysts (which could potentially yield a viable organism) must be destroyed to obtain these select cells, has led to controversy in some segments of society. Part of the controversy derives from stating that ES cells came from embryos. Others consider the fertilized egg to have the same rights as born humans, and so the designation would not be relevant for them. This controversy has helped spawn techniques designed to transfer the nucleus to an egg with inhibitors of trophoblast development, so that no implantable blastocyst would develop; or of an adult cell into preexisting ES cells, as well as obtaining ES cells from the 8-cell morula stage, mimicking preimplantation genetic diagnosis that leaves the 7 cells capable of blastocyst development. Other types of pluripotent stem cells include gonad precursors found in fetal tissue called embryonic germ cells (another misnomer, embryonic cells from a fetus), and embryonic carcinoma cells (e.g., teratocarcinomas), which can produce all cell types, but in a highly disorganized manner. The last major class of stem cells, multipotent stem cells, gives rise to a limited number of cell types. These cells can be tissue (e.g., mesenchymal stem cells, skin stem cells, or blood stem cells) or organ-specific cells (neural stem cells). Multipotent stem cells can be found in most organs of the body and are responsible for organ growth and maintenance. The best-characterized multipotent stem cells are the hema - topoietic stem cells (HSCs), which are responsible for the continuous production of blood cells. The relative ease of isolating HSCs, as well as a wealth of in vitro and in vivo assays, has allowed hematopoiesis to become an important model system for the study of stem cell biology (57, 58). The prospective isolation of HSCs required development of assays for all blood clonal outcomes, of monoclonal antibodies that subdivide the marrow, and of high-speed cell sorters. (For a description of a cocktail of antibodies to purify HSCs, see ref. 43 and references therein.) NEUROSURGERY VOLUME 65 NUMBER 2 AUGUST

4 CHESHIER ET AL. The advancement of monoclonal antibody technology coupled with fluorescence-activated cell sorting in the 1980s allowed scientists to characterize hematopoietic cells based on expression of specific cell surface proteins indicated by fluorescently labeled monoclonal antibodies against these proteins (18). For example, specific combinations of cell surface molecules could be used to isolate T cells, B cells, and myeloid cells. In 1988, using a panel of negatively selecting monoclonal antibodies that stain mature blood cells (lineage lo or neg), coupled with 2 other positively selecting antibodies recognizing cell surface proteins that could enrich BM for HSC activity (Thy1.1 and Sca-1), Spangrude et al. (87) utilized fluorescence-activated cell sorting to isolate the first pure hematopoietic multipotent stem and progenitor cell population from mouse BM. Later experiments showed the most primitive population of HSCs (the longterm [LT] HSCs) could be isolated to homogeneity by the addition of 2 makers: strict Lineage cells that simultaneously express high levels of the tyrosine-kinase stem cell factor receptor, c-kit (60). The end result of these experiments was determination that the Lineage, Sca-1 +, c-kit +, Thy1.1 lo expressing cells were the only cells in the BM capable of giving rise to LT myeloid-erythroid and lymphoid cells when transplanted into irradiated mice. Thus, a predetermined combination of cell surface marker antibody staining (c-kit +, Lineage, Sca-1 +, Thy1.1 lo ) could be used to isolate pure populations of LT-HSCs from whole BM. This result allowed for direct analysis of these cells, rather than an indirect analysis based on functional outcomes in transplanted animals. For instance, early studies by researchers such as Till and McCulloch certainly implied the existence of HSCs, yet the cells were never analyzed directly. The prospective isolation of HSCs, and their multipotent/oligopotent progenitors (single cells that produce all or few blood cell types, but do not self-renew) revealed that all stem cells are not equal in that some cells can produce progeny almost indefinitely ( true stem cells), whereas other cells are more limited in their selfrenewal capacity and are more restricted in the types of cells they can develop into (progenitor cells) (Fig. 3). The studies purifying HSCs provided the foundation for later experiments in which pure HSCs and blood cell lineage-specific progenitors were subsequently isolated in both mice and humans (83). The isolation of each cell type of blood (stem cells, restricted progenitors, mature cells) has allowed scientists to ascertain the hierarchical organization of the hematopoietic system based on proliferation, differentiation, and self-renewal, and identify important other cell types and signaling factors essential for the process of self-renewal and commitment (Fig. 3). Such hierarchical organization exists in solid organ systems, including the CNS (Fig. 1) (7, 15, 23, 24, 33, 35, 46, 71, 79). The successful balance of self-renewal, proliferation, and differentiation requires a high degree of fidelity, and disturbances in the organization can lead to disease. NEURAL STEM CELLS Until recently, it was unclear if the principal hierarchical organization of the hematopoietic system applied to the CNS. In the hematopoietic system, multipotent stem cells capable of extensive proliferation and self-renewal give rise to a series of progressively more lineage-restricted progenitors with less proliferative capacity (Fig. 3). There is now growing evidence that the CNS has a cellular organization based on self-renewal and differentiation similar to hematopoiesis (Fig. 1). The central and historical dogma of CNS biology was that little if any turnover occurs within the CNS of mature vertebrates, especially in terms of new neuronal growth. This supposition was first challenged by a study demonstrating that the hormonally responsive growth of the hyperstriatum ventrale, pars caudalis in canaries was in part attributable to new neuronal growth derived from a rapidly cycling progenitor cell population located within the subventricular zone (34). More recently, long-term cultures of human and rodent CNS tissues have revealed the existence of cells as a population capable of maintaining the continuous production of neurons, oligodendrocytes, and astrocytes; in some studies, they contained retroviral tagged clonal precursors of all 3 lineages (17, 56, 73, 75, 99). Indeed, several groups have devised methods for the isolation of such cells from patients (102). This technique has great potential for obtaining, harvesting, manipulating, and transplantation of these cells into patients. Although these progenitor/stem cells can grow in monolayers, their most distinctive in vitro characteristic is their ability to form round clusters of selfadherent cells termed neurospheres (75, 99). Single cells derived from neurospheres could produce both neurons and glia when transferred into rodent brains. These in vivo experiments demonstrated that a clonal population of CNS cells derived from neurospheres in vitro contained neural tissue-specific progenitor/stem cells (75, 93, 99). In the adult human brain, neurospheres containing CNS stem cells have now been isolated from the periventricular area of the forebrain lateral ventricles (subventricular zone) and the dentate gyrus of the hippocampus (6, 24). Neurospheres have also been derived from adult rats and human fetuses (6, 31, 46, 71, 82, 96, 97). Uchida et al. (93) achieved the first prospective isolation of human CNS stem cells when they applied the experimental principles of HSC isolation to the characterization of cell suspensions made from the fetal subventricular zone. Fluorescenceactivated cell sorting was used to separate cells that had the specific cell surface marker combination: CD133 +, 5E12 +, CD24 neg/lo, CD45 neg (blood cell lineage), and CD34 neg (blood vessel lineage and itinerant HSCs). Neurospheres initiated from single clones of sorted cells of the above phenotype were able to form new neurospheres in culture, and could differentiate into neurons, astrocytes, and oligodendrocytes (trilineage differentiation) in vitro. The same cells demonstrated engraftment, migration, and differentiation when transplanted into the brains of newborn immunocompromised mice (88, 93). Putative CNS stem cells based on the above phenotype have already demonstrated potential clinical utility. These cells can halt the formation of neurological defects when injected into the brains of a mutant mice strain that normally develop lysosomal storage diseases (51) of the CNS such Batten disease (unpublished observations), improve motor activity when injected into trau- 240 VOLUME 65 NUMBER 2 AUGUST

5 A NEUROSURGEON S GUIDE TO STEM CELLS FIGURE 3. A wealth of information from the study of hematopoietic stem cells suggests that an intricate cellular hierarchy exists whereby a hematopoietic stem cell commits to lineage-restricted stem cells and eventually to the various cell types of the blood. Within the bone marrow, support cells, such matically damaged spinal cords of mice (20), and migrate and produce new neurons in ischemic rodent brain (49). Using different combinations of cell surface markers and fluorescentprotein reporter constructs, fluorescence-activated cell sorting has also been used to isolate CNS stem cell populations from mice (16, 52, 76). The prospective isolation of CNS stem cells will greatly expand our knowledge of both normal and abnormal CNS development. The development of CNS stem cell biology may lead to a better understanding of lineage-restricted CNS progenitors and the potential for therapeutic use. Undoubtedly, knowledge gained from this endeavor, a better ability to manipulate and dictate the fate of progenitor cells into coher- NEUROSURGERY as the stromal cells and pericytes, produce necessary signaling environments that allow for both the maintenance of hematopoietic stem cells and their differentiation to myeloid and lymphoid cell types. ent, functional neurological units and knowledge of migratory patterns of normal NSCs, and the ability for effective delivery of therapeutic genes to human neurological malignancies will greatly expand the neuro-clinician s arsenal to combat devastating diseases of the CNS. The identification of CNS stem cells may also provide insights into the development of primary brain tumors. CANCER STEM CELLS Cancer biology remains one of the most intensely studied areas of scientific discourse. Although basic research has led to a wealth of information regarding the molecular mechanisms VOLUME 65 NUMBER 2 AUGUST

6 CHESHIER ET AL. responsible for the transformation of normal cells into cancers, our concept of the cellular biology of neoplasms has remained poorly illuminated. The cellular constitu - ents of solid neoplasms in - clude both tumor cells and non-tumor stromal elements such as blood vessels, fibro - blasts, hematopoietic cells, and nontransformed cells from the tissue of origin. Until recently, the neoplastic tumor cells were assumed to be a relatively homogeneous group of cells, each capable of producing more cancer cells within the tumor and as well as producing metastases. Investi gations of the clonality of cancers verified that most neoplasms result from the malignant transformation of a single cell into a tumor cell (26, 27, 63). This hypothesis holds that the transformed cell could simply replicate, producing new tumor cells with relatively equivalent biological properties. Evidence in support of this model is abundant in both retinoblastoma and colon cancer where the accumulation of sequential mutations in the cell cycle machinery causes cancerous growth. This standard model of cancer formation was further reinforced by the intensive utilization of tumor cell lines in the context of cancer research. However, several experimental results conflicted with the predictions of the standard model. Investi gations have shown that single cancer cells did not grow uniformly well in cell culture, cancer cells were not equally sensitive to therapeutic agents both in vitro and in vivo, and large numbers of cancer cells were usually required to produce tumors in transplant animal models. Although it is possible that the variability among cancer cells in these studies resulted from inefficiencies of the assays, the observations also suggest that there are intrinsic differences between individual tumor cells. The differences seen among cancer cells may be explained in part by the principles of CNS stem cell biology. The overlap between stem cell biology and cancer biology is striking. Both tissue stem cells and cancer cells share the ability to proliferate, self-renew, and give rise to differentiated progeny. In the case of the tissue stem cell, the balance between differentiation and proliferation must be highly regulated (Fig. 4, left). Under normal conditions, neural stem cells residing adjacent to vascular endothelium differentiate to produce neuronal and glial progeny. In the case of cancer, there is marked FIGURE 4. The vascular niche hypothesis proposes that in addition to the subventricular zone and the subgranular zone of the hippocampus, stem cells are housed adjacent to endothelial progenitor cells (EPCs). In the local signaling environments, or niches, produced by the endothelial cells, neural stem cells are capable of asymmetric division to produce other neural stem cells as well as committed progeny that eventually produce neurons, astrocytes, and oligodendrocytes. The process of self-renewal and commitment are tightly regulated and ensure that neoplastic growth does not take place (left side). The tumor literature supports the existence of a similar vascular niche in brain tumors. Cancer stem cells have been observed to reside in close association with endothelial cells where they are likely to make use of the same signaling pathways that maintain normal stem cells (right side). deregulation of the balance between proliferation and differentiation, resulting in uncontrolled tumor growth and incomplete differentiation (Fig. 4, right). Cancer stem cells, similarly housed adjacent to endothelial cells, proliferate and produce both other cancer stem cells and non-cancer stem cell tumor cells. The leaky nature of blood vessels in a tumor allows for an abundance of inflammatory and BM-derived progenitor cells within the mass of the tumor. In addition, and almost predictably, cancer and stem cells share many molecular mechanisms mediating these complex processes (100, 101). For instance, the cell cycle machinery, self-renewal genes, and growth factors necessary for normal stem cell existence are the same genes utilized by many cancer cells. Furthermore, both stem cells and cancer cells can be transplanted into animal models recapitulating normal stem cell regenerative function in the case of stem cells and tumor development with cancer cells. The prominent similarities between cancer and normal stem cells have led to the development of the Cancer Stem Cell Hypothesis as proposed by Reya et al. (74). This hypothesis consists of 2 complementary and non mutually exclusive components. The first component postulates that normal tissue stem cells are the target for transforming mutations and successive insults result in the eventual formation of a tumor. The second component is that within every cancer there exists a specific subset of cells that continuously give rise to all the other 242 VOLUME 65 NUMBER 2 AUGUST

7 A NEUROSURGEON S GUIDE TO STEM CELLS cancer cells. This subset of tumor cells are the cancer stem cells and are the only cells within a tumor that possess the ability to self-renew, continuously proliferate, and can give rise to metastases. Although some tumor cells may have some proliferative capability, only the cancer stem cell can reproduce itself and another nonreplicating tumor cell. Furthermore, because tumors are clonogenic, all of the heterogeneity in a tumor is produced by the cancer stem cells. Although some of the tumor cell diversity can result from progressive mutagenesis, there is ample evidence supporting that the heterogeneity within cancers reflects aspects of normal differentiation processes, which would be expected if cancer stem cells are giving rise to differentiated progeny (28, 29, 39). In other words, the histobiological properties of a cancer tend to reflect its tissue of origin because the cancer stem cell and its progeny continue to recapitulate the phenotype of the original tissue, although in a disorganized and unproductive manner. Thus, a tumor can be viewed as a dysfunctional organ system, in which the cancer stem cell gives rise to phenotypically diverse (albeit dysfunctional) progeny that have limited proliferative potential and no ability for self-renewal. It should be noted that recent studies by Morrison et al. (72) question some of the most important components of the cancer stem cell hypothesis. Using melanoma as a model, the group led by Morrison showed that nearly 1 in 4 cells possessed proliferative ability and spawned cancer. Although the final verdict has yet to be determined, it is likely that the cancer stem cell hypothesis is applicable to some tumors and not to others, such as melanoma. Mutations Accumulating in Normal Stem Cells Lead to Cancer A well-established concept of cancer biology is that over time a series of genetic mutations leads to the malignant transformation of a normal cell into a cancer cell. These mutations result in the disruption of the cellular machinery controlling the cell cycle, cell growth and inhibition, apoptosis, immune surveillance, genomic stability, differentiation, and self-renewal (65, 100). Mutations can propagate only if a cell divides, thus in cellular terms, time is synonymous with proliferative events, and mutations occur in the context of cell divisions occurring frequently during development and more slowly in a mature organism. This in part explains why most cancers cluster in the pediatric group (in which more cells are dividing per unit time) or the geriatric group (in which slowly dividing cells have been undergoing divisions for a long period of time). The idea that normal tissue stem cells accumulate genetic mutations leading to cancer formation came about as a direct extension of studies describing the cellular organization of hematopoietic cells. In both fetal and adult tissues of mice and humans, only LT-HSCs are capable of the continued production of myeloid-erythroid cells (60 62). Unlike LT-HSCs that have a lifespan measured in years, the immediate progenitors of HSCs, short-term HSCs, myeloid restricted progenitors, and lymphoid restricted progenitors, only have lifespans of weeks to months (62). Thus, only LT-HSCs possess the proliferative capacity and lifespan necessary to accumulate the threshold of mutations leading to cancer. Put more simply, the longevity of the tissue stem cell also increases the risk and exposure of the cell to transforming mutations. Experimental evidence for this idea was noted in animal models of leukemia in which mice in pre-leukemic states could transfer leukemia only when HSCs were transplanted into irradiated hosts (59). Miyamoto et al. (59) postulated that if LT-HSCs were harboring mutations, then leukemic patients in complete remission should have mutations in their remaining HSCs because the more committed short-term HSCs are derived from the more primitive LT-HSCs. These investigators analyzed the LT-HSCs from Hiroshima survivors who developed acute myelogenous leukemia (AML) and were in long-term remission. They found that a significant fraction of LT-HSCs in these patients continued to harbor the AML pathoneumonic AML1- ETO translocation mutation, but because other mutations promoting deregulated growth were not present in these patients, they remained in remission. The mutated LT-HSC was indistinguishable from the LT-HSC without the translocation and displayed no evidence of deregulated growth in vitro. It should be noted that, despite the proposed necessity of mutation accumulation in normal stem cells for cancer formation, it is very possible that a transforming event (or events) can occur in a more mature progenitor downstream of the long-lived stem cells, or that the effects of the mutations that accumulate in the stem cells are manifested in a downstream progenitor. This final transformation of a progenitor cell has experimental evidence in mouse and human leukemogenesis and neuroepithelial tumor formation (40, 41, 44, 45, 91, 111). There is also experimental evidence suggesting that genetic alterations within lymphocytes can lead to mouse T cell leukemia independently from HSCs (110). However, this result is not inconsistent with the stem cell hypothesis because, upon antigen stimulation, lymphocytes regain the stem cell properties of proliferation and selfrenewal. Thus, a lymphocyte can be viewed as a unipotent stem cell capable of giving rise to progeny that can be used in an immune reaction or to produce new lymphocytes with different antigen binding properties through the process of affinity maturation. The idea of stem cells as the focal point of cancer mutations is a striking contrast to more traditional models in which any cell can be subject to mutations leading to cancer. However, it is more parsimonious to postulate that a cell possessing the unique ability for self-renewal and extensive proliferation is better suited to forming a tumor than a terminally differentiated cell lacking these properties. Cancer Stem Cells Establish and Propagate Tumors The second component of the cancer stem cell theory postulates that a specific subset of tumor cells, the cancer stem cells, are the only cells capable of the continued production of tumor cells (74). Despite the assumed homogeneity of cancer cells in the standard model of cancer cell organization, the reality of tumors is quite different. Histologically, tumor cells can display a large variation in appearance, often resembling the spectrum of differentiated cell types of the tissue from which the tumor arises. Also, only a very limited number of cancer cells from NEUROSURGERY VOLUME 65 NUMBER 2 AUGUST

8 CHESHIER ET AL. hematopoietic and solid malignancies can grow in vitro or in vivo in animal transplant models (28, 29, 36, 37, 39, 86). Investigations have shown that, in the case of hematopoietic and some solid malignancies, only 1 in 100 to 1 in primary tumor cells are capable of reproducing the tumor in vivo (4, 5, 14, 60). The standard model would postulate that all cancer cells are equivalent, and that each cell has a low probability of proliferating in the setting of the experimental conditions. The cancer stem cell theory would posit that only the cancer stem cell subset can give rise to new cells, and because this population is rare, their activity on a per cell basis is rare. The cancer stem cell hypothesis predicts that prospectively isolating these rare subsets of cells from the non-stem cell cancer cell would result in markedly different cellular proliferation. This stem cell subset should be highly enriched for in vivo and in vitro tumor growth, and the non-stem cell fraction should be highly depleted of these abilities. The experimental confirmation of the cancer stem cell hypothesis was first achieved by Bonnet and Dick (14) for AML. They postulated that because cancer stem cells are derived from normal stem cells, they should have similar cell surface phenotypes. Using a panel of monoclonal antibodies used to prospectively isolate normal human HSCs, they determined that patient-derived AML cells with the cell surface combination of CD34 +, CD38, Lineage were the only cells capable of transferring disease to immunocompromised mice. These AML stem cells represented only a small yet variable fraction of all tumor cells. Later investigations demonstrated that the AML stem cells were CD90, which differs from normal human HSCs that are CD90 + (1, 2). Thus, the AML stem cell had an overlapping, but distinct, cell surface phenotype compared with its normal counterpart. A growing number of investigators have isolated cancer stem cells from a number of leukemias including chronic myelogenous leukemia, blast crisis chronic myelogenous leukemia, and myeloproliferative disorders (30, 42, 45). In each case, the cancer stem cell demonstrates the unique property of serial transplantation compared with the non-cancer stem cells derived from the same tumor. In solid tumors, the presence of cancer stem cells was confirmed when Al-Hajj et al. (4, 5) prospectively isolated a rare subset of human breast cancer cells with the cell surface phenotype CD44 +, CD24 neg/lo, Lineage. Cells with this phenotype were the only cells capable of forming new tumors upon injection into the mammary glands of immunocompromised mice. Since then, a growing number of mouse and human solid tumors have been fractionated on the basis of cell surface marker expression, and the existence of cancer stem cells confirmed experimentally including human neuroepithelial tumors, head and neck squamous cell carcinomas, and colon cancer (19, 21, 53, 70, 85). BRAIN TUMOR STEM CELLS The cancer stem cells relevant to a neurosurgical practice are those that initiate tumors of neuroepithelial origin. Bailey and Cushing (8) were the first to articulate the concept of brain tumor arising from a progenitor cell. They proposed that tumors were derived from progenitor cells residing in the periventricular zone. The first experimental evidence suggesting the existence of brain tumor stem cells (BTSC) was published by Hemmati et al. in 2003 (38), although data dating back to the 1980s had suggested the existence of such cells (77, 78). It is known that brain tumor cells can grow in clusters in defined media containing epithelial growth factor and fibro - blast growth factor-2. These specialized clusters are referred to as tumorspheres (TS) and these culture-derived spheres are very similar to in vitro sphere formation with normal CNS stem cells, meaning they contain a conglomerate of stem and progenitor cells with committed neurons, glia, and oligodendrocytes. The formation of a tumor or neurosphere is a culture phenomenon, but represents an interesting mechanism for creating differential signaling environments allowing for specialization of cells within the sphere. Hemmati et al. demonstrated that only a rare fraction of cells cultured from medulloblastoma and ependymoma could form TSs. Furthermore, cells from TSs could give rise to more spheres in culture and were capable of forming tumors when transplanted into the brains of immunocompromised mice. The investigation demonstrated that only a rare subpopulation of cells within these tumors were capable of proliferation and self-renewal. These properties along with tumor engraftment with in vivo transplantation form the hallmark characteristics of cancer stem cells. Similar experiments by other investigators confirmed the presence of a rare subset of TSs initiating glioblastoma multiforme (GBM) cells that were also capable of tumor formation upon xenograft transplantation (32, 92, 109). A special cell population with tumor-forming ability (i.e., tumorigenicity) has also been prospectively isolated from glioma cell lines (50, 85). C6 glioma cells growing in vitro contain a rare subset of cells that can be separated by fluorescenceactivated cell sorting on the basis of the cell s ability to efflux Hoechst, the vital deoxyribonucleic acid staining dye (50). The Hoechst effluxing cells, termed side population, seemed to contain the in vitro and in vivo growth potential of the cell line. This information suggests that a cancer stem cell or progenitor is also active in a well-established tumor cell line. Later experiments confirmed that the dye exclusion ability in the glioma cell line was attributable to the presence of multidrug resistance transporters on the cell surface (66). This is an interesting finding given that multidrug resistance is known to be highly expressed in other normal tissue stem cell populations, including HSCs (13, 25, 94). The identification of a cell surface marker, which could prospectively isolate cancer stem cell containing populations from primary tumor specimens, was first achieved by Singh et al. (84, 85) in a series of experiments published in Singh et al. used the normal CNS stem cell surface antigen, CD133, to fractionate fresh medulloblastoma and GBM specimens. The investigation demonstrated that CD133 + expression was measured in a minority of tumor cells, but were also highly enriched for the ability to produce TSs in vitro (84). Subsequent experiments demonstrated that these CD133 + tumor cells were the 244 VOLUME 65 NUMBER 2 AUGUST

9 A NEUROSURGEON S GUIDE TO STEM CELLS only cells capable of forming new tumors when implanted into the brains of immunocompromised mice (85). Singh et al. showed that as few as 100 CD133 + cells were capable of forming tumors in vivo, whereas CD133 cells did not result in tumor formation in vivo. CD133 + has also been recently used to isolate BTSCs from TSs derived from ependymoma cultures (89). These studies represent the first significant experimental evidence of possible BTSCs. Further experimentation is required to confirm these results and subsequent studies are needed to then fully characterize the tumor stem cell. Thus far, the results are based on the utilization of only a single cell surface marker, CD133, which is also shared by HSCs and normal tissue CNS stem cells. However, no stem cell population has been prospectively isolated to relative homogeneity on the basis of a single cell surface marker. In fact, neither the experiment by Singh et al. nor any subsequent study utilizing CD133 as a marker for BTSCs included an in vivo limit dilution experiment comparing sorted cell populations with whole tumor, which is currently the only method able to ascertain the relative purity of any given sorted population. In general, multiple markers are required to purify tissue stem cells. In the case of HSCs, 10 to 15 separate monoclonal antibodies are needed to isolate a pure stem cell population, and in the case of human fetal CNS stem cells, a combination of at least 4 antibodies has been necessary to isolate a relatively homogeneous stem cell population (61, 93). The need for cell purity is not a trivial matter, as this homogeneity is critical for the accurate determination of gene and protein expression patterns specific for BTSCs. In the case of leukemia whereby the data are strong and multiple markers are available, it is possible to isolate culprit stem cells to purity. Because of the lack of reproducible markers, identification of stem cells for brain tumors, most notably GBM, has been very difficult. In these cases in which markers are not available, larger cell numbers including non-cancer stem cells, have been injected to reproduce growth of the tumor. With identification of other markers, it will be possible to purify brain tumor initiating stem cells and reduce input numbers necessary to generate tumors in xenografts. These BTSC-specific gene and protein profiles will be the foundation for the development of BTSC-specific therapies as well as help determine the relationship of BTSCs to their normal CNS stem cell counterparts. Using a different marker, Ogden et al. (64) separated human GBM on the basis of cell surface marker A2B5 and CD133. The key finding of this study was to note that tumor cell formation segregated into the A2B5 + fraction. However, in their study, both A2B5 + CD133 + and A2B5 + CD133 cells could give rise to tumors in a potent manner when transplanted into the brains of immunocompromised host mice. Several groups have questioned the validity of CD133 as a BTSC marker (12, 47). Further - more, our own analysis of patient GBM and medulloblastoma cell surface protein expression revealed that a significant number of these cells do not express detectable levels CD133 protein (unpublished results). It could be hypothesized that cell surface antigens associated with tumor stem cell markers should be TABLE 1. Properties of neural stem cells and cancer stem cells a Neural stem cells Cancer stem cells Cell surface marker CD133 CD133; A2B5 Self-renewal Unlimited Unlimited Proliferation Low Variable Location SVZ, SGZ of hippocampus Variable Proximity to vessel Adjacent Adjacent Signaling pathway regulating fate EGF, bfgf, Wnt, Shh, TGFβ EGF, bfgf, PDGF, Wnt, Shh, TGFβ Chemosensitivity Sensitive Variable with some resistance Radiation Sensitive Variable with some resistance a SVZ, subventricular zone; SGZ, subgranular zone; EGF, epidermal growth factor; bfgf, basic fibroblast growth factor; Shh, sonic hedgehog; TGF, transforming growth factor. measurably expressed across the majority of patient specimens. Furthermore, in vivo analysis of CD133 + and CD133 cells derived from adult GBM tumors demonstrated growth in both populations, and at least 2 instances wherein the CD133 cells formed large tumors in a mouse xenotransplant model (unpublished observations, Weissman laboratory). We are not suggesting that CD133 is not a potential BTSC marker, but we stipulate that CD133 may be a reliable marker within a tumor sample, but not necessarily between tumor samples. A more durable marker should be able to prospectively isolate BTSCs across the majority of brain tumor samples. At this time, the sum of all experiments represents only a relatively few number of tumor samples, thus more experiments with careful designs and controls are needed to discover multiple markers that can be utilized to prospectively isolate BTSCs. Since the discovery of BTSCs, a number of laboratories have investigated mechanisms specifically regulating the biology of these cells as opposed to whole tumors. CNS stem cells and BTSCs share many similarities (Table 1). Similar to normal CNS stem cells, BTSCs reside next to blood vessels (vascular niche hypothesis) (95). The study of normal stem cells has implicated members of Wnt, sonic hedgehog, and transforming growth factor-β as important regulators of stem cell growth and development (11, 55). Interestingly, the vascular niche has been shown to produce many of these factors (95). A recent study from the Hopkins group has shown the importance of the transforming growth factor-β family member bone morphogenetic protein to the regulation of BTSCs (69). They demonstrated that activation of bone morphogenetic protein 4 greatly inhibits the growth of tumors via its direct effect on reducing BTSC growth. In a different experiment, abrogation of the sonic hedgehog pathway, an important developmental signaling pathway, greatly reduced the ability of GBM tumor spheres to NEUROSURGERY VOLUME 65 NUMBER 2 AUGUST

10 CHESHIER ET AL. grow tumor in vivo, and greatly reduced the sphere-forming ability of CD133 + GBM cells (10). Sonic hedgehog is implicated in maintaining cells in an undifferentiated state, and molecules capable of blocking this signal can potentially induce differentiation of the BTSCs. Similarly, the Wnt signaling pathway has been shown to be an important regulator of neural stem cell self-renewal (48) and may serve a similar role in brain tumors. Furthermore, just as normal stem cells are relatively resistant to the effects of irradiation and chemotherapy, BTSCs seem to possess similar properties. For instance, Rich et al. (9) demonstrated that CD133 + GBM cells were highly radio resistant and this resistance was mediated by the up-regulation of DNA repair mechanisms. Also, the ability of GBM tumor spheres to grow in vitro and in vivo was inhibited to a lesser degree than control cells when exposed to a number of standard chemotherapeutic agents including temozolomide (54, 77). Is cancerous growth the result of proliferation of a stem cell or a more committed progenitor? In most systems, it is not clear whether the culprit involved in oncogenesis is a stem cell or a progenitor cell. Part of the problem arises from the fact that the definitions of a stem and progenitor cell are functional; the lack of markers allowing for the prospective isolation of the normal and cancerous CNS stem cell further complicates studies of the stem versus progenitor origin of brain tumors. In the case of medulloblastoma, 2 recent articles (80, 107) illustrate that accumulation of mutations within a specific progenitor is responsible for the formation and progression of this tumor. The authors used labeling experiments to mark specific Olig2 progenitor populations and showed that accumulation of mutations within this and only this progenitor population results in medulloblastoma formation. It is important to note that evidence in other systems is sparse at best. It is possible that in some tumors accumulation of mutations in a progenitor causes tumor formation, whereas in others mutations in the parent stem cell are the cause; the jury is still out on this topic. These findings underscore the importance of the types of data that can be derived from the study of brain cancers from a stem cell perspective. WHY EVERY NEUROSURGEON SHOULD CARE ABOUT BTSCS Despite advances in the molecular biology of cancer, surgical technique, radiotherapy, and chemotherapy, limited progress has been made in reducing the mortality associated with CNS tumors. The historic model of cancer holds that most tumor cells are relatively homogeneous with respect to proliferative and self-renewal potentials. The cancer stem cell theory suggests that this model is inaccurate and this may account for some failures in cancer treatment. Specifically, current treatment modalities were designed to measure success by reductions in tumor bulk and volume; however, in this approach, there is no way to confirm that the cancer stem cells, if present, are being eliminated. Assuming that the cancer stem cell theory is accurate, then debulking tumors is a less fruitful task than the identification and elimination of the BTSCs in guaranteeing the elimination of the malignancy. The prospective isolation of BTSCs will provide better targets for the development of new therapies and to new ways to measure treatment efficacy. New agents could target the molecular mechanisms supporting the growth of these cells rather than the confusing multitude of pathways present within normal cells or the bulk of nonproliferating tumor cells. For example, prospective isolation of BTSCs may allow for design of pharmaceuticals targeting cancer stem cell specific signaling pathways (108). In addition, gene and protein array analysis (22, 68, 81), as well as high-throughput screening of biological and synthesized molecules, could lead to small molecule and immune-based therapies (67, 98) directed specifically against unique BTSC antigens. Efforts using array and immune-based technologies are already in preclinical trials for hematologic malignancies. Given the devastating nature of neurological malignancies, the interval between isolation of pure BTSCs and phase I trials will hopefully be short. Currently, new techniques in molecular imaging and biomarkers in combination with imaging protocols are beginning to help quantify and locate BTSCs before, during, and after treatment (103). What is the role of the neurosurgeon in the setting of these potential BTSC-specific therapies? Neurosurgeons are currently at the forefront of the research being performed to isolate and characterize these cells. The collection of patient tumor samples is the first step in any study attempting to identify BTSCs, and the role of the interested neurosurgeon in this process is invaluable. The surgical technique undertaken will help determine the integrity of the tumor specimen and thus ultimately the quality and quantity of potential BTSCs. Surgery will remain the mainstay of initial treatment of malignant neuroepithelial tumors for the foreseeable future, but the development of potential local therapies directed against residual BTSCs will also fall within the neurosurgeon s realm. The ability to rapidly characterize the gene and protein profiles of tumors and perhaps the isolation of tumor stem cells has great potential to yield patientspecific therapies. Future therapies could be based on the response seen for individual and patient-specific BTSC testing when screened against conventional and experimental agents. The field of cancer and stem cell biology will no doubt contribute to improved understanding of disease and potential new treatments. An exciting future awaits neurosurgeons as practices evolve into a combination of surgical and cell therapeutics, resulting in better outcomes for patients with brain cancer. Disclosure Samuel H. Cheshier, M.D., Ph.D., is a fellow of the Giannini Foundation and a Van Wagner Fellow. M. Yashar S. Kalani, M.D., Ph.D., is a fellow of the Paul & Daisy Soros Foundation, the Hanbery Society, and of the Howard Hughes Medical Institute. Steven L. Huhn, M.D., is an investigator at Stem Cells, Inc. Irving L. Weissman, M.D., is a consultant for Stem Cells, Inc. REFERENCES 1. Ailles LE, Gerhard B, Hogge DE: Detection and characterization of primitive malignant and normal progenitors in patients with acute myelogenous leukemia using long-term coculture with supportive feeder layers and cytokines. Blood 90: , VOLUME 65 NUMBER 2 AUGUST