TOXICOLOGICAL SCIENCES 120(S1), S269–S289 (2011) doi:10.1093/toxsci/kfq370 Advance Access publication December 16, 2010

Stem Cells in Toxicology: Fundamental Biology and Practical Considerations Kyung-Sun Kang* and James E. Trosko†,1 *Adult Stem Cell Research Center, Laboratory for Stem Cell and Tumor Biology, Department of Veterinary Public Health, College of Veterinary Medicine, Seoul National University, Sillim-Dong, Seoul 151-742, Korea; and †Center for Integrative Toxicology, Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, East Lansing, Michigan 48824 1

To whom correspondence should be addressed at Center for Integrative Toxicology, Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, 246 Food Safety and Toxicology Building, East Lansing, MI 48824. Fax: (517) 432-6340. E-mail: [email protected]. Received August 30, 2010; accepted November 28, 2010

This ‘‘Commentary’’ has examined the use of human stem cells for detection of toxicities of physical, chemical, and biological toxins/toxicants in response to the challenge posed by the NRC Report, ‘‘Toxicity Testing in the 21st Century: A vision and Strategy.’’ Before widespread application of the use of human embryonic, pluripotent, ‘‘iPS,’’ or adult stem cells be considered, the basic characterization of stem cell biology should be undertaken. Because no in vitro system can mimic all factors that influence cells in vivo (individual genetic, gender, developmental, immunological and diurnal states; niche conditions; complex intercellular interactions between stem, progenitor, terminal differentiated cells, and the signaling from extracellular matrices, oxygen tensions, etc.), attempts should be made to use both embryonic and adult stem cells, grown in three dimension under ‘‘niche-like’’ conditions. Because many toxins and toxicants work by ‘‘epigenetic’’ mechanisms and that epigenetic mechanisms play important roles in regulating gene expression and in the pathogenesis of many human diseases, epigenetic toxicity must be incorporated in toxicity testing. Because modulation of gap junctional intercellular communication by epigenetic agents plays a major role in homeostatic regulation of both stem and progenitor cells in normal tissues, the modulation of this biological process by both endogenous and endogenous chemicals should be incorporated as an end point to monitor for potential toxicities or chemo-preventive attributes. In addition, modulation of quantity, as well as the quality, of stem cells should be considered as potential source of a chemical’s toxic potential in affecting any stem cell–based pathology, such as cancer. Key Words: adult human stem cells; epigenetic toxicology; gap junctions; Barker hypothesis; three-dimensional in vitro cultures.

Historically, the field of toxicology has employed multiple scientific disciplines to identify both the basic mechanisms of physical, chemical, and biological agents that could induce, acutely or chronically, any harmful effect on a target organism or ecosystem. Multiple end points of toxicity, at the molecular, biochemical, cellular, physiological, pathologies at the whole organism or ecosystem levels, morbidity, mortality, etc., have been used. Specific concepts and techniques (in vitro/in vivo, epidemiological, etc.) have been used to identify specific mechanisms of toxicities (e.g., mutagenesis, cytotoxicity, and altered gene expression or ‘‘epigenetic toxicity’’) that might be responsible for the pathogenesis of diseases. This science of toxicology, having both a basic science or academic component and a real-life practical application to understand and prevent life-threatening crises, related to accidental exposures, overdoses, suicide attempts, deliberate poisoning, unintended consequences of mixtures of toxins and toxicants or of mismatches of genetic, gender, developmental stage factors, species differences, etc., has provided a solid foundation of knowledge to date. However, as has been highlighted in the NAS Report on Toxicity testing in the 21st Century: A Vision and a Strategy (Committee on Toxicity Testing and Assessment of Environmental Agents, 2007) provides a framework for this ‘‘Commentary.’’ Essentially, the report stated, in unequivocal terms, what was obvious, namely, the current concepts and testing procedures were not sufficiently adequate for extrapolation to human beings. This is not to say that previous molecular, in vitro, animal experimental models, epidemiological studies, and computer modeling did not contribute some useful information to the potential efficacy or safety of new chemicals/pharmaceuticals. Rather, the Report pointed out that these approaches were not very precise and that improvements had to be made. In

Ó The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected]

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addition, other practical concerns were raised, such as the cost of developing new drugs, insensitivities of the tests to predict side effects, differential developmental stage responses, interactions of mixtures of chemicals/drugs/endogenous factors and even interactions with microbial indigenous organisms (Flier and Mekalanos, 2009; Niwa et al., 2010; Phillpott and Girardin, 2010; Turnbaugh et al., 2009), gender and individual genetic differences. Consequently, given the limitations of the factors stated in that NRC Report, and recognizing up-front, there will be builtin limitations of any new strategy or technique to bridge the theoretical and practical gaps between any surrogate in vitro human test system and the specific extrapolation of a predicted harmful health effect of an exogenous agent to either a specific individual or to a population. Included in these limitations to use new molecular, biochemical, or cellular end points is the fact that the individual whole organism can never be fully represented in any in vitro assay. Currently, to mimic the immune system, each individual organ response and organ-to-organ response and the diurnal effects in vitro have yet to be developed. Unique genetic and phenotypic backgrounds, representing specific inherited DNA sequences and modified epigenetic expression of that DNA, gender differences in coupling with toxin/toxicant exposures, developmental state responses, nutritional and dietary modifications of exposures, life style behaviors, and constant exposures to multiple potential mixtures of modifying chemicals, are not easily incorporated in a single in vitro assay. At this point, the challenges to improve the current imperfect approaches being used might seem daunting. However, this challenge to the field of toxicology comes at a time when new exciting advances are being made in the field of stem cell biology. It will be the objective of this Commentary to explore how advances in this exploding field might be utilized to offer a potential partial solution to the challenge posed by the NRC Report. CAN HUMAN STEM CELLS BE USED AS A NEW TOOL IN TOXICOLOGY? THE NEED TO UNDERSTAND BASIC STEM CELL BIOLOGY.

Although it is very tempting to jump directly into this field to use human stem cells to assess the potential of any toxin/ toxicant to affect stem cells, as it is currently being done, it might be wise to examine some theoretical and practical matters related to our current state of understanding basic stem cell biology and their roles in normal development and any stem cell–related disease. Although the concept of stem cells has long been a focus of intellectual examination in the fields of embryology, developmental biology, plant biology, and cancer research, arguably, the cloning of Dolly (Wilmut et al., 1997) and the isolation of human embryonic stem cells (Shamblott et al., 1998; Thomson et al., 1998) have brought stem cells into the public’s and scientific/medical communities’ consciousness. Clearly, the theoretical possibility of applying human stem

cells for ameliorating many human diseases seemed possible before attaining any fundamental understanding of the complex factors which control a stem cell’s behavior. However, at the moment of trying to identify the realistic potentials from unrealistic hype about the stem cells, attempts are being made to apply stem cells for wide diverse applications, such as for toxicity testing. With so little being known about the regulation of the genes of a stem cell, it could be a major problem to ensure its proper behavior when put to its many potential uses. To date, stem cells have been considered to be used for (1) basic mechanistic understanding of how a stem cell regulates the genome and its cell behavior (cell proliferation in a symmetrical or asymmetrical fashion, differentiation, apoptosis, immortality, senescence, etc.), (2) regenerative medicine or stem cell therapy, (3) drug discovery, (4) toxicity testing of pharmaceuticals and stem cell therapy, (5) genetic therapy, and (6) the role of stem cells in stem cell–derived diseases and in the aging process. It would be impossible to examine the potentials and a limitation of all these potential applications, at a time when there is a blizzard of publications appearing as this overview is being written. Consequently, we will limit our examination on the use of stem cells in the various branches of toxicology. In all likelihood, there might never be a technical approach that can overcome the complexities of understanding, let alone of controlling how agents entering the human system, from conception to the geriatric stage of our life. It cannot be stated strongly enough that, in many cases, the assays used had no mechanistic bases for the toxicity end point (Trosko and Upham, 2005). Equally important, our understanding of the pathogenesis of many human diseases is not known. Consequently, trying to predict the pathogenesis of a human disease (or how to prevent or cure the disease) by using data, derived from inadequate toxicity testing, only describes the inadequacies of our current state of knowledge. Probably the best example of this dilemma was the introduction of the concept, ‘‘carcinogen as mutagen’’ and the development of the bacterial assay to detect chemical ‘‘mutagen/carcinogens’’ (Ames et al., 1973). Today, although the pathogenesis of cancer is still not completely understood, we do know it is more than mutagenesis and the bacterial assay clearly is inadequate to detect all agents that can contribute to the multistage, multi-mechanism processes of carcinogenesis (Pitot and Dragon, 1991; Weinstein et al., 1984). UNDERSTANDING HUMAN DEVELOPMENT IS TO UNDERSTAND STEM CELL BIOLOGY

From the moment of conception, the fertilized egg (‘‘totipotent’’ stem cell), which contains the total unique genome of that prospective individual, must now face the complex interactions of the external environment’s affect on the pregnant female, of the genetic background of that mother, of her nutritional status and diet, of her life style behavior, of her psychological stress level, and of any medical treatments. From that dynamic interaction, the implantation of the totipotent

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stem cell starts a concatenation of events to commit this stem cell to form the embryonic or pluripotent stem cell (Markert, 1984). As these cells appear in the blastocyst, they, in turn, change the microenvironment causing a further restriction of these pluripotent stem cells to become, multipotent stem cells that are committed to specific organ types. In turn, even more microenvironment changes occur as the lineage terminally differentiated cells appear from the multipotent stem cells. Bipolar stem cells, such as the oval cells of the liver appear (Coleman et al., 1997; Muller-Borer et al., 2004). Finally, progenitor stem cells, such as those seen in the lymphohematopoietic system appear. Progenitor or transit-amplifying cells, or those derived from a stem cell are, somehow, committed to a finite life span. The terminally differentiated cell, such as a red blood cell or a lens cell, is restricted from proliferating. In effect, the embryonic stem cell is the pluripotent stem cell, whereas adult stem cells are usually restricted or committed to fewer lineage-type differentiated cells in the context of an adult body. Operationally, a stem cell is defined as a cell that can divide either symmetrically or asymmetrically after being given an appropriate external signal. Although it is still unknown what might be the mechanism for controlling whether a stem cell divides, symmetrically or asymmetrically, empirically some growth conditions do influence this cellular decision, including specific growth factors (Kawase et al., 2004), or substrates, such as feeder layers or extracellular matrices (Chaiswing and Oberley, 2010; Comoglio et al., 2003; Garamszegi et al., 2010) or O2 levels and antioxidants (Csete, 2005; Go and Jones, 2010; Lavrentieva et al., 2010; Lengner et al., 2010; Lin et al., 2005; Linning et al., 2004; Zachar et al., 2010). One speculation relates to these factors being able to restrict the stem cells from expressing their connexin genes or from having functional gap junctional communication, as well as controlling the cell division plane, which could create the situation where the two new daughter cells are either binding to the same niche substrate or only one is and the other is freed from substrate restricted differentiation (Trosko et al., 2000). Because neither daughter directly communicated with each other via gap junctions, the one daughter now has a different set of signals to suppress its Oct4 gene and express its connexin genes and starts to differentiate (Fig. 1). In addition, as will be discussed later, the operational definition of a pluripotent, embryonic, or induced pluripotent stem (iPS) cell includes their ability to form teratomas when placed back into an adult recipient. Fundamentally, it appears that there are several critical factors that help to maintain a stem cell as a stem cell. The first is the ‘‘niche’’ or microenvironment that controls the cell physiology and gene expression for the appropriate stem cell state (Juliano, 2002; Li and Neaves, 2006; Simsek et al., 2010; Tumbar et al., 2004; Voog and Jones, 2010). This includes the specific extracellular matrices (Fuchs et al., 2004; Ott et al., 2008). In addition, it is well documented that the state of oxygen tension is

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important in maintaining the state of stemness (Csete, 2005; Lengner et al., 2010; Pervaiz et al., 2010; Zachar et al., 2010). Although the specific hormones, growth factors, endogenous cytokines, and nutrients have yet to be identified for each stem cell in vivo for each organ, they, too, can influence intracellular signaling that affect the genes regulating ‘‘stemness’’ (Asselin Labat et al., 2010; Joshi et al., 2010; Kubota et al., 2004). Furthermore, specific ‘‘master genes’’ (Holden, 2006) and stemness genes, such as Oct4, a POU-transcription factor, are found as universal markers for not only embryonic stem cells (Nichols et al., 1998; Niwa et al., 2000; Okamoto et al., 1990) but a variety of normal adult stem cells (Chang et al., 2004; Kim et al., 2009b; Lin et al., 2007; Linning et al., 2004; Neupane et al., 2008; Tai et al., 2005). In addition, to use stem cells in vitro as a potential surrogate of what might happen to that stem cell in vivo when exposed to a potential toxin/toxicant, one cannot ignore diurnal factors that might be happening in vivo that might not be happening in vitro. Another gene, the gap junction gene or the connexin gene, whose functions have been attributed to both growth control and differentiation (Lo, 1996; Loewenstein and Kanno, 1966; Yamasaki and Naus, 1996), were shown in several organisms to be expressed only after ‘‘compaction’’ stage in the early blastocyst (Lo and Gilula, 1979). In many adult stem cells, neither the connexin genes are expressed nor are the gap junctions functional (Chang et al., 1987, 2004; Kao et al., 1995; Kim et al., 2009; Lin et al., 2007; Linning et al., 2004; Matic et al., 1997, 2002; Tai et al., 2003; Trosko et al., 2000; Yang et al., 2007). This might suggest that the Oct4 stemness gene must suppress, directly or indirectly, the connexin or differentiation-dependent genes. In particular, this is a relevant observation, in that the reason stem cells normally require a feeder layer is because they not only need the factors supplied by the nonproliferative feeder layer cells but they are not ‘‘contact inhibited,’’ as are cells with functional gap junctions. This was seen in the isolated of human kidney adult stem cells in 1987, as were cancer cells, which, also, are characterized by no functional gap junctions (Chang et al., 1987). THE DYNAMIC, HOMOESTATIC NATURE OF NORMAL DEVELOPMENT, AND HEALTH

In using human stem cells for potential toxicity testing, one must keep in mind the dynamic nature of stem cell biology in vivo so that any in vitro result will not be misinterpreted in any extrapolation to potential toxicities in vivo. From a single fertilized egg to the fully developed human, approximately 100 trillion cells are present, with an estimated 200þ cell types within the organism. Conceptually, this developmental process, involving an undifferentiated totipotent stem cell, with the total genetic information to produce a conscious human being, involves many complex interactions and organization of molecules, biochemical reactions, organelle formation within cells, that organize into tissues and organisms, which form

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FIG. 1. These diagrams illustrate two possible means by which stem cells (Oct4þ) decide to divide by symmetrical or asymmetrical division. In panel (A), if a stem cell binds to a specific extracellular matrix, as symbolized by attaching to a coated plastic dish, the signal received combines with signals in the medium (growth factors, Caþþ, nutrients, oxygen, etc.) to stimulate genes and gene products to bring about the division plane to be perpendicular to the attachment plane. As a result, both daughter cells continue to have identical signaling as did the maternal stem cell (symmetrical cell division or expansion of the stem cell population). In panel (B), the stem cell binds to a different substrate molecule, as represented by a natural extracellular molecule, such as laminin or collagen type 4. In this case, the signal this substrate molecule induces a different intracellular signal that interacts with the same signals from the medium, Caþþ, oxygen, etc., to stimulate different genes and gene products to cause a division plane within the stem cell to be formed parallel to the attachment plane. In this case, the daughter cell on the bottom will mimic the same intracellular signaling as its maternal stem cell. Important to note that if these stem cells do not have functional GJIC, then these signals are not transmitted to the other daughter cell because that daughter cell does not interact with the substrate signal. As a result, these daughter cells receive a different set of combined signals that trigger a commitment to become a progenitor and ultimately terminally differentiated progeny.

organ systems to eventually create a conscious human being. All this takes place via unique environmental (physical, chemical, biological) interactions with the unique set of genes. Philosophers of biology view the human being as the result of the ‘‘hierarchical principle’’ (Brody, 1973; Von Bertalanffy and Novikoff, 1945), where the organization of each basic subunit (atoms) can lead to molecules, which have ‘‘emergent’’ properties not found in the individual element of the subunit. Molecules organize to form biochemical reactions/functions and structures, such as specific organelles, which, in turn, help to form specific cell types. The organization of specific cell types helps to form tissues and organs, having specific functions that can interact, in a cybernetic fashion

(Potter, 1974; Trosko, 1998; Weiner and Schade, 1965), to form, ultimately, a conscious human being. It is critical to point out that this dynamic development from the single fertilized egg or totipotent cell requires a delicate coordination of communication from the external environment on cell to activate specific genes, which, in turn, alters the phenotypes of cells. This, then, alters the microenvironment of a colony of cells (blastocyst). Consequently, even more changes now occur to create even more phenotypes to influence more gene changes (‘‘epigenetic’’ changes) to cause another sequential set of phenotypic changes. This description of development has been beautifully characterized by Markert (1984).

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Recognizing that the development of a complex multicellular human being, during the evolution from a single cell organism, had to involve acquisition of a unique set of genes, besides sharing those genes that are needed to survive in an environment requiring oxygen for generating energy and for surviving in a physical environment of external radiation, appropriate temperature, and a given amount of gravity. That evolutionary transition created new phenotypes, such as ‘‘growth control,’’ cellular differentiation to produce specific functions needed for various survival strategies (muscles for movement, neurons for coordinated muscle movement, sensing, and consciousness; etc.), programmed cell death or apoptosis for systematic removal of un-necessary cells at specific stages of development, and senescence (Trosko, 2007a). Although it can be argued that this evolutionary transition (Revel, 1988) involved many new genes, it is interest that the early multicellular organ acquired a family of genes, the connexin genes (Cruciani and Mikalsen, 2005; Willecke et al., 2002), whose proteins, selforganize to form a six-element membrane-associated hemichannel, the connexon (Evans and Martin, 2002). Connexons from two adjacent cells can form a gap junction, through which ions and small regulatory molecules can freely pass to act as a ‘‘sink’’ or ‘‘source’’ (Sheridan, 1987), to help synchronize electrotonic or metabolic functions (Fig. 2). To underscore the unique importance of gap junctional intercellular communication (GJIC) for the evolution of the metazoan, one needs only to examine a cancer cell, a cell that has lost growth control, is unable to terminally differentiate, cannot apoptose normally, and has lost ‘‘mortality’’. Because the normal cells seem to govern growth control via ‘‘contact inhibition’’ (Eagle, 1965) and given that the cancer cell has been described has having lost ‘‘contact inhibition’’ (Borek and

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Sachs, 1966), and as referring to a cancer as a ‘‘disease of differentiation’’ (Markert, 1968), a ‘‘stem cell disease’’ (Greaves, 1986; Pierce 1974; Till, 1982], or as ‘‘ontogeny as partially block ontogeny’’ (Potter, 1978), it might not be too surprising, as Loewenstein and Kanno (1966) first demonstrated, cancer cells are characterized as having dysfunctional GJIC. The fundamental role that these gap junctions play in basic cellular functions, such as growth control (Loewenstein 1966), differentiation and development (Houghton, 2005; Lo, 1996; Wei et al., 2004), and apoptosis (Wilson et al., 2000), also illustrated that, when compared with the normal rat bipolar stem cell, in the same cells, transfected with a dominant negative connexin43 gene (Upham et al., 2003), no organized structure is formed. When these dysfunctional rat liver cells are transplanted into the syngeneic rats, they will eventually form small liver tumors over many months. The same rat liver cells, transfected with the Ha-ras oncogene, also, have dysfunctional intercellular communication (de Feijter et al., 1990), but quickly form tumors. Although both type of cells lack normal GJIC, one has only a dysfunctional connexon structure to inhibit transmission of important signals (D/N Cx43 cells), whereas the other (Ha-ras cells) has an oncogene that sends two signals to block connexons by hyperphosphorylation of the connexin protein and to signal mitogenesis. This illustrates that the inhibition of GJIC is a necessary, but insufficient, component for the cancer process. Additional evidence has been provided showing that GJIC can be modulated, either reversibly or stably, by most, if not all, tumor-promoting chemicals’ GJIC (Budunova and Williams, 1994; Trosko and Chang, 1988). Growth factors, hormones, extracellular matrix, and cytokines can also block

FIG. 2. A cell-to-cell channel is formed by connexins, which first oligomerize as hexamers (now called connexons or hemi-channels), transported to the cell surface, and dock with connexons in the adjacent cells. A gap junction is formed when several channels cluster at one particular spot. A gap junction may be composed of channels formed of more than one type of connexin. Thus, the basic structure of the channel has remained unchanged since its earlier inception.

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GJIC (El-Sabban et al., 2003; Kielan and Esen, 2004; Matesic et al., 1996; Murray and Fletcher, 1984; Ren et al., 1994; Rudkin et al., 1996; Shiokawa-Sawada, et al., 1997; van Zoelen and Tertoolen, 1991). Antisense connexins in normal cells can induce a tumorigenic phenotype (Goldberg et al., 1994; Ruch et al., 1995) and that many oncogenes can stably inhibit GJIC (Trosko and Ruch, 1998). On the other hand, transfection of tumor cells with the normal connexin genes can reduce the tumorigenicity of the cells (Eghbali et al., 1991; Mehta et al., 1991; Rose et al., 1993; Hirschi et al., 1996; Jou et al., 1993; Mesnil et al., 1995; Naus et al., 1992; Zhu et al., 1991). Transfection of tumor cells with tumor suppressor gene can increase GJIC (de Feijter-Rupp et al., 1998). Prevention of the downregulation of GJIC by tumor-promoting chemicals can be accomplished by treatment with chemopreventive agents, such as retinoids, carotenoids, green tea components, ginsenosides, epicatechin, resveratrol, caffeic acid, cocoa polyphenols, pigenin, lycopene, Quercetin, tangeretin, etc. (Chaumontet et al., 1994; Kang et al., 2000; Kim et al., 2009; Lee et al., 2010a,b; Livny et al., 2002; Mehta et al., 1986, 1989; Na et al., 2000; Nakamura et al., 2005a, 2005b; Nielson et al., 2000; Ruch et al., 1989; Sai et al. 2001; Stahl and Sies, 1998; Stahl et al., 2000; Upham et al., 2007; Zhang et al., 1992). Some chemotherapeutic and anticancer drugs, such as SAHA (Ogawa et al., 2005) and Lovastatin (Ruch et al. 1993), can increase the expression of the Cx43 gene. Most importantly, to demonstrate a broader influence on homeostatic prevention of many different diseases that the various members of the family of connexin genes can play, it has now been demonstrated that there exists not only a number of knockout connexin mice with different disease syndromes (Willecke et al., 2002) but also, a number of human inherited connexin mutation have been identified, such as the CharcotMarie-Tooth syndrome, zonular pulverulent, Oculodentodigital dysplasia, Erythrokeratodermia variabilis, and Clouston syndrome (Abrams and Bennett, 2000; Kelsell et al., 2001; MartinNieto and Villalobo, 1997; White and Paul, 1999). This, then, should set the state for highlighting the fact that gap junctions exist in every human organ. Their regulation during early development is critical for normal cell number control, the proper cell types needed in specific organs, and their normal homeostatic control. Even during adolescent sexual development, homeostatic functioning of each mature organ and the normal aging process, maintaining GJIC is critical. To reiterate, to ignore GJIC in the development of any human stem cell in vitro toxicology assay is to ignore one of the potential mechanisms by which a given chemical toxin/ toxicant might lead to some disease endpoint or to some efficacious pharmaceutical use. The importance of this family of genes only added to the forms of cell communication mechanisms found in single cells, namely primitive extracellular secreted signaling molecules (Hardman et al., 1998; Le Rothe et al., 1980) and coordinated (and often cross-talking) intracellular signaling pathways, that

are triggered by the extracellular signaling factors. The gap junctional intercellular communication mechanism now, for the first time, linked the extracellar endogenous communication factors, which represent thousands of entities (secreted factors, such as hormones, cytokines, chemokines, growth factors, nutrients, pressure, and tension on cell membranes) (Discher et al., 2009; Holst et al., 2010; Nelson et al., 2006; Pardanaud and Eichmann, 2009; Saunders et al., 2001), specific extracellular matrices (Wondimu et al., 2006), and an almost infinite number of exogenous chemicals to a finite number of intracellar signaling pathways. These pathways can activate or inactivate extant proteins in the cell and activate or inactive transcription factors to regulate specific genes that control proliferation, differentiation, apoptosis, stress responses, or senescence. In brief, the extracellular communication or ‘‘stromal-epithelial–type’’ interactions trigger intracellar communication signals to modulate GJIC between either homologous or heterologous cells within tissues (Barcellos-Hoff, 2001, 2005, Rizki and Bissell, 2004). In tissues, this complex set of signals must be viewed as between the few adult stem cells, their transit-amplifying or progenitor daughters, and the terminally differentiated offspring (Fig. 3). This insight might suggest the use of organ-specific adult stem cells, grown in 3D, such that the committed organ-specific stem cell creates its own unique microenvironment (its own secreted extracellular matrix, specific differentiated lineage cells which provide normal homeostatic control signals). Disruption of this microenvironment, the ability of the organ-specific stem cell to control proliferation, differentiation, and apoptosis by some agent might be one indication of its potential toxicity. The inclusion of this coordinated set of communication mechanisms, as a part of the homeostatic regulation of normal development from the embryo, fetus, neonate, adolescent, mature, and geriatric human being, is important in understanding the development of a new approach using human stem cells for toxicity testing. Disruption of any of these communication mechanisms is a part of both an adaptive response of cells in a multicellular organism and, potentially, of a nonadaptive consequence. To disrupt any of these three mechanisms during critical periods of embryonic, fetal, and neonatal development, when organ systems are being determined, could lead to either embryonic or fetal lethality, as well as teratogenesis. Attempts have already been used to use embryonic stem cells for developmental toxicity testing (Augustine-Rauch et al., 2010; Buesen et al., 2009, Chapin and Stedman, 2010; van Dartel et al., 2010), as well as for toxicities of known carcinogens (Lin et al., 2010a). Yet programmed disruption of cell-cell communication is required for inhibiting ‘‘contact inhibition’’ controlling growth or tissue regeneration or wound healing (Stein et al., 1992; Yancey et al., 1979). Therefore, either nonprogrammed or chronic modulation (increased or decreased) of GJIC can be considered as potentially toxic biological effect on homeostatic control (Rosenkranz et al., 2000; Trosko et al., 2002; Upham and Trosko, 2009).

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FIG. 3. Gap junctions in cellular homeostasis. Extracellular signals, such as growth factors, toxicants, extracellular matrices, and cell adhesion molecules that vary for each cell type (adult stem cell, progenitor and terminally-differentiated), interact with membrane receptors, which then activate intracellular signal transduction pathways that induce the transcription of genes (A–C) through activated transcription factors. These specific intracellular pathways operate under cascading systems that cross-communicate with each other in controlling the expression of genes that direct the proliferation, differentiation, and apoptosis of cells within a tissue. These multiple intracellular signaling checkpoints are further modulated by intercellular signals traversing gap junctions, thereby maintaining the homeostatic state of a tissue. Abnormal interruption of these integrated signaling pathways by food-related and environmental toxicants results in diseased states, such as the tumor promotion phase of carcinogenesis, teratogenesis during early development, atherogenesis, immunotoxicity, reproductive toxicity, and neurotoxicity.

TOXICITY END POINTS AT THE CELL LEVEL: THE ROLE OF STEM CELLS IN CARCINOGENIC TESTING

Exposure of the human organism to physical, chemical, biological agents, and even psychological stress will eventually be biologically translated into some response at the cell level: Potter (1973) stated this succinctly: ‘‘The cancer problem is not merely a cell problem; it is a problem of cell interaction, not only within tissues, but also with distal cells in other tissues. But in stressing the whole organism, we must also remember that the integration of normal cells with the welfare of the whole organism is brought about by molecular messages acting on molecular receptors.’’ It seems that three potential consequences to this exposure will be (1) mutation, caused by either an error in DNA repair or by an error of DNA replication (genotoxicity), (2) cell death by necrosis or apoptosis (cytotoxicity), and (3) altered gene expression at the transcriptional, translational, or posttranslational levels (epigenetic toxicity). Various molecular and in vitro assays have been used in the past, primarily for genotoxicity and cytotoxicity assessments. Arguably, in view of the scores of published studies that have been used to determine which assay or combinations of assays should be used for toxicity assessments, no consensus has been achieved. In fact, it might be said, in large part, it was the reason the NRC Report (Committee on Toxicity Testing and Assessment of Environmental Agents, 2007) was published. The results of

these assays were (1) not consistent, (2) did not measure what they were supposed to measure, (3) generated data from abnormal cells, (4) extracted from cell types not relevant to the human system, (5) derived from the wrong conditions for measuring toxicities that occur in vivo at different conditions, and (6) in many cases, were misinterpreted (Trosko and Upham, 2005). Although in vitro assays are being developed to measure ‘‘epigenetic toxicants’’ or, as some refer to them as ‘‘nongenotoxicants,’’ they, too, carry many of the same limitations of those other in vitro assays. There should be no doubt that endogenous- and exogenousinduced intracellular signaling mechanisms were evolutionarily designed to regulate, differentially, gene expression. The concept of epigenetics is about the development and homeostatic differential regulation of the total genome (Meissner, 2010; Mohammad and Baylin, 2010). On the other hand, genetic or environmentally induced abnormal expression of genes is the basis of epigenetic toxicity (Feinberg, 2010; Portela and Esteller, 2010). However, the recent microarray technologies, sophisticated as they are, still leave much to be desired. Clearly, patterns of altered gene expression from exposed or diseased tissues can be reproduced and even have useful applications. However, as will be illustrated later, extracting molecular expressed genes from normal, treated, or diseased tissue is extracting messages from a heterogeneous mixture of cell types, for example, a few adult stem cells; many progenitor or transit-amplifying cells and the terminally

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differentiated cells. In addition, some of these cells are stressed; others are dying of apoptosis, some are senesced, others might be mutated, and others are invading cells. Therefore, the total message examined is the net expression of all these types of cells, each with a different gene expression pattern. This point has been specifically addressed (Debey et al., 2004; Freezor et al., 2004; Shen-Orr et al., 2010), as they stated: ‘‘Traditional microarrays’’ can neither distinguish between variations in gene expression resulting from an actual physiological change versus differences in cell-type frequency nor identify the contributions of different cell types to the total measured gene expression. Another important point needs to be highlighted, namely although ionizing and ultraviolet light radiation can induce chromosome and point gene mutation, respectively, they can, at low non-cell killing doses, induce oxidative stress, which, in turn, can altered gene expression. In effect, at these non-cell killing doses, they can have epigenetic effects (Trosko, 2000; Trosko and Suzuki, 2009; Upham and Trosko, 2009). It should also be pointed out that, at killing doses, any agent (radiation, chemicals, biological) can be an indirect epigenetic toxicant, in that the released substances from cell killing can act to stimulate the surviving cells to wound healing. Of course, toxicity, in vivo, involves the tissue’s response to the exogenous chemical or therapeutic stem cell or stem cell– derived differentiated daughters, as well as to the consequences of the chemical or stem cell products on the immune system (Baldridge et al., 2010; Essers et al., 2010; Maggini et al., 2010). Therefore, toxicity must be viewed in a larger perspective. As the foreign agent (chemical or stem cell product) enters the body, it will directly or indirectly interact with (1) the three types of cells (adult stem cells, transit amplifying cells, and the terminally differentiated cells) in the tissue and (2) with cells of the immune system. In both cases, intracellular signaling occurs. In the case of the cells of the immune system, various bioactive secreted factors are released that now can interact with the epithelial/endothelial cells that might have also been ‘‘primed’’ by the same toxicant (Fig. 4). Determination of how exogenous agents, for example, chemicals or stem cell products, might induce specific intracellular signaling to trigger epigenetic changes in the targeted tissue, in vivo, will be the challenge. If the cellular consequence of the altered gene expression is to cause contactinhibited, nonmitotic cells to proliferate, GJIC can be assumed to be downregulated. The short-term consequence could be wound healing. The long-term consequences of chronic, inflammatory downregulation of GJIC, for example by IL-6, or TGF-a, would be hyperplasia or tumor promotion (Trosko and Tai, 2006). One of the characteristic properties of this class of agents that block gap junction function is also to inhibit apoptosis, in that tumorpromoting chemicals block apoptosis (Bursch et al., 1984; Schulte-Hermann et al., 2000). It has been noted in mesenchymal stem cell transplantation studies, alteration in the apoptotic rate of cells was noted (Sun et al., 2009).

On the other hand, if the agent causes either cells of the immune system and the cells of the affected tissues to upregulate GJIC, cells could differentiate or apoptose. Now, as it might relate to stem cell therapy and tests to predict some aspects of safety related to transplanting stem cells into adult organisms, the stem cell, itself, will be subject to in vivo endogenous microenvironment factors that could lead to enhanced symmetrical cell division (proliferation), induced asymmetrical cell division (differentiation, apoptosis), or senescence. These cells also can induce a modulated immune response by their own secreted factors that could illicit a cellular response in the targeted tissue (Ne´meth et al., 2009; Zhao et al., 2010). This type of unexpected response has been reported (Lee et al., 2010c; Thirabanjasak et al., 2010).

CRITICAL FACTORS INFLUENCING THE LIMITATIONS OF CURRENT IN VITRO ASSAYS

Although it was quite understandable that the introduction of a quick, easy, and inexpensive bacterial assay to screen for potential mutagenic agents in the expensive drug efficacy and safety protocols, bacterial cells simply are not human cells, whose functions go far beyond just proliferating or cell survival. For the use of in vitro mammalian cells, most assays have used either immortalized or cancer cells, again, for convenience of use. However, it should be obvious that abnormal cells, grown on plastic or glass, in log phase and in two dimensions (2D), under 20% oxygen, with artificial media, have little resemblance to the complex in vivo milieu. In addition, without an immunological or dynamic physiological mimicking culture environment, these abnormal cells had little chance of predicting how any of the three classes of normal human cells (the adult stem cells, the progenitor or terminally differentiated cells) would react in vivo. In vivo, these three types of cells are all communicating between themselves via stromal-epithelial interactions, communicating with the extracellular matrix and usually in 3D in stationary phase at various levels of oxygen tension and with potential interactions with other physiological factors and the immune system. In the normal tissue, each cell type (the few stem cells, the many progenitor and differentiated cells) is physiologically different. Consequently, regardless of the toxic potential of any agent, upon contact with each cell type, there will be a differential response. Assuming the stem cell, by being characterized as a ‘‘primitive, undifferentiated cell,’’ might not express various metabolizing enzymes needed to deal with a toxic chemical. In fact, there is mounting evidence that these stem cells express drug transporter genes that pump out various classes of toxic chemicals (Doyle and Ross, 2003; Martin et al., 2004; Shimano et al., 2003; Zhou et al., 2001). This might be one potential limitation for the use of pure stem cell populations to test for genotoxic chemicals. Although the field of characterizing DNA repair of stem cells, as compared with

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FIG. 4. The diagram tries to incorporate a ‘‘systems’’ aspect of how a physical, chemical, or biological agent could affect a multicellular organism. At noncytotoxic concentrations or doses, an agent could simultaneously trigger oxidative stress in both the cells of the immune tissues and the epithelial/endothelial/ stromal cells in various organs. Upon induction of reactive oxygen species and of oxidative stress and induction of intracellular signaling in various cell types of the complex immune system, various cytokines would interact on tissues, containing the three fundamental cell types (adult stem cells, progenitor, and terminally differentiated cells). Given that these cells would have been exposed to the toxic agent and that they, also, would have reacted to the agent differentially because of their different physiological/phenotypic state, the interaction of all three types could be very different (e.g., the normal stem cells might be induced to proliferate asymmetrically, any initiated pre-cancerous stem cell might proliferate symmetrically, the progenitor cells might be induced to proliferate symmetrically and to migrate, as in wound healing, and the terminally differentiated cell might adaptively respond or to apoptose) in response to the inflammatory signal. In summary, each cell type of the immune system and of the various organ tissues, with their different expressed genes and cellular physiology, will respond differently to sublethal exposure to agents inducing oxidative stress–triggered intracellular signaling and epigenetic alterations. The interaction of inflammatory agents on preexposed organ cells could be an additive effect, a synergistic response or possibly, even an antagonistic effect. This could explain the wide range of diseases in which the inflammatory process seems to play a prominent role.

their progenitor and differentiated linage cells, has yet to be done in a systemic fashion for human stem cells, it is critical that this type of information be generated (Tsunoda et al., 2010; Ropolo et al., 2010). At this stage, one cannot assume that DNA repair systems are equivalent in the lineage-derived and terminally differentiated daughter cells. Nor can one assume that all stem cells in each tissue are equivalent in their ability to survive a toxic insult. This has been demonstrated in the sensitivity of small intestinal versus the large intestinal stem cells in vivo (Potten, 1989; Potten et al., 1994). In addition, in an intact human being, exposure to any agent, which interacts with both the cells of the immune system and cells of the solid tissues, affects both cell types directly. Cells of the immune system are induced to release a complex set of highly reactive molecules. At the same time, that same agent

has interacted, directly, with all three cell types in solid tissues (in the epithelial, endothelial, or fibroblast cell pools), sending signals in each to alter their phenotypes. Now the immune cells’ released factors interact on the ‘‘primed’’ epithelial, endothelial, or fibroblast cells. That kind of complexity of reactions will be hard to be completely mimicked in vitro (Lee et al., 2010; Maggini et al., 2010; Monneret, 2009; Zhao et al., 2010). Where does that leave the future of toxicity testing? Because it is both practically and ethically impossible to test new drugs, any commercial chemical or therapeutic stem cell product, directly, on human beings, we only find out, after its long-term use on thousands or millions of human beings, the potential risk, caused by incomplete understanding of the mechanisms that could lead to individual genetic sensitivities or drug interactions with either other endogenous factors or exogenous

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chemicals. The concept of ‘‘personalized medicine’’ or ‘‘personalized therapy’’ has been coined to take into account part of this problem (Collins, 2010; Khoury et al., 2010). The lack of testing of the growing list and widespread use of the socalled ‘‘natural remedies’’ is of a similar concern. The development of 3D ‘‘organoids,’’ generated from various types of normal human stem cells, appears to ‘‘bridge the gap’’ (Claude et al. 2010; Guguen-Guillouza et al., 2010; Pampaloni et al., 2007; Smalley et al., 2006), seems to be the only option we have (Trosko and Chang, 2010). Of course, previous and current attempts have been made to use 3D in vitro cultures for embryotoxicity and teratogenicity. The historic use of neurospheres, other 3D organoids, and ‘‘micromass’’ systems illustrate the awareness that there might be stem cells in these population-based in vitro systems and these 3D structures start to mimic the in vivo tissue situation. Yet, their use, at a time of uncertain mechanisms of toxicities at are taking place in these systems, real differences between rodent and embryonic and organ-specific stem cells, nonvalidated and universally accepted markers for stem cells, limits the ability to accurately extrapolate to the human situation. One might imagine, given all the limitations even this approach will have, that a practical strategy for a protocol might be to utilize a battery of an adult human stem cells from a finite number of major organs from a human male and a female (lung, heart, muscle, brain, liver, kidney, lung, eye, etc.). These would be tested as they are induced to form 3D structures, in which most of the major cell types are found, together with the specific normal extracellular matrices found in the normal organ from which that stem cell was obtained. To deal with the potential interaction of the immune system or of other physiological factors, these 3D-specific stem cell–derived structures could be cocultured via various insert culture devices or flow-throw devices. At this point, one must consider what must be the source of the human stem cells. Finally, illustrating the importance of mimicking a tissue-like microenvironment is the study by Tsunoda et al. (2010), which showed that an activated KRAS gene inhibited normal cell polarity and apoptosis, as well as expression of DNA repair–related tumor suppressor genes, in 3D, but not 2D, in vitro culture of human colonic cells.

DOES THE TYPE OF HUMAN STEM CELL MAKE ANY DIFFERENCE FOR USE IN TOXICITY TESTING?

Since the early days of stem cell research, an explosion in stem cell technology research has and continues to occur. The majority of these studies deal with (1) exploring basic stem cell biology, (2) trying to by pass ethical issues related to creating human embryonic stem cell lines, (3) exploring new strategies for creating pluripotent stem cell lines, (4) understanding and maximizing growth and differentiation conditions for stem cells, and (5) developing conditions for potential therapeutic

usages. There have even been a few attempts to consider their use for drug discovery and safety assessment or toxicity testing (Davila et al., 2004; Guguen-Guillouza et al., 2010; van Dartel et al., 2010; Vogel, 2005), and now an explosion of bioengineering of various scaffolds, substrates, geometry, and matrices are being developed for stem cell therapy (Buzanska et al., 2010; Kilian et al., 2010; Ruiz et al., 2008a, 2008b). From the beginning, the isolation of embryonic human stem cells, while opening up many potential applications, are well known to have formidable technical scientific and medical limitations, besides the ethical ones. The first of which is that the operational definition is that these embryonic or pluripotent stem cells will form teratomas when placed back into an adult organism (Fig. 5). The attempt to form somatic cell nuclear transplant (SCNT) pluripotent stem cells (Byrne et al., 2007), while trying to circumvent the ethical problems, never really panned out (Cibelli, 2007). However, recent reports suggest that this SCNT approach can have its efficiency increased (Inoue et al., 2010). With the remarkable report that one could create an iPS cell by introducing a finite number of embryonic genes, such as Oct4, sox2, etc., into differentiated fibroblasts and a few other somatic differentiated cells, opened up a floodgate of observations of multiple ways of creating ‘‘iPS-like cells’’ (Baker, 2010; Takahashi and Yamanaka, 2006). Even the use of small molecules or appropriate cell culturing conditions could bring about the ‘‘reprogramming’’ of a somatic cell into an ‘‘iPSlike pluripotent’’ cell (Baker, 2010; Emre et al, 2007; Jia et al., 2010; Lin et al., 2010; Nagy and Nagy 2010; Warren et al., 2010). As this field is actively being developed at the time of the writing of this Commentary, it would be difficult to predict their potential to be used, successfully, compared with ‘‘normal’’ human embryonic and organ-specific adult human stem cells. That some abnormalities have been detected in these iPS cells should raise cautionary applications (Kim et al., 2010; Polo et al., 2010). At this stage, it has to be pointed out that, once the transformation to an iPS state has occurred, these cells, also, by operational definition, are capable of forming teratomas if placed back into an adult organism. Some have argued that this ‘‘problem’’ should be placed in the risk/benefit category of concern, if for no other reason, even if a teratoma does form, it is a relatively easy tumor to treat. It has to be compared with the potential benefit of a therapeutic consequence of stem cell treatment. However, other genetic or epigenetic alterations, especially when various viral or molecular vectors are used, could, by insertional mutagenesis, lead to serious technical and toxicological problems (Dolgin, 2010; Hu, et al., 2010). Although embryonic stem cells have the potential to produce all the differentiated cell types of the adult human being, and while several induced embryonic stem cell–derived differentiated cells have been characterized, not all cell types have yet been created in vitro. Nor has there been any validated study to demonstrate that these cells might response identically to

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FIG. 5. H9 Human Es cells colony (left) and teratoma formation (right).

toxicants as their counterparts naturally produced in vivo. It has to be pointed out, also, that cultures of populations of embryonic stem cells are not homogeneous (Pera, 2010; Stewart et al., 2010). The discovery of human cord blood and other multipotent mesenchymal stem cells (MSCs), and neural stem cells has opened up an addition strategy for both toxicity testing and new drug screening (Trosko and Chang 2010). Multipotent stem cells (MSCs), first reported as bone-forming progenitors, can differentiate into mesodermal lineages including adipocytes, osteocytes, and chondrocytes (Fig. 6). Furthermore, MSCs can also differentiate into endodermal and ectodermal lineages (Lee et al. 2004; Pittenger et al., 1999). These characteristics indicate the potential of MSCs in cell-based clinical strategies. MSCs can

be isolated from various adult tissues including umbilical cord blood, bone marrow, adipose tissue, periosteum, trabecular bone, synovium, skeletal muscle, and deciduous teeth (Barry and Murphy 2004). Among these, human umbilical cord bloodderived MSCs exhibit higher potencies of proliferation and differentiation. The use of umbilical cord blood, which is routinely discarded as waste material in the delivery room, would be advantageous because it is an abundant source of MSCs and is obtainable by noninvasive means. By using MSCs for the drug development and toxicity testing, because MSCs can differentiate into various lineage cells, the effects of toxicants or drugs can tested or screened during differentiation. For example, in anti-obesity drugs

FIG. 6. Human umbilical cord blood-derived mesenchymal stem cells can differentiate into three germ-layered cells (ectoderm, mesoderm, endodermal cells).

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development, MSCs’ differentiation ability into adipocytes could be used to test whether any chemicals or toxicants can affect to these processes. In senescence studies, aging of an organism is a complex process that involves the decline of multiple systems that are required to maintain the homeostasis of cells and tissues in addition to the impairment of tissue maintenance and repair (Kirkwood, 2005; Trosko, 2003a, 2007b). Recent speculations suggest that an age-associated decline in adult stem cell function might also be associated with these processes. (Trosko, 2008c). Modification of chromatin by epigenetic regulatory mechanisms, including acetylation, deacetylation, and methylation of histones (Mohammad and Baylin, 2010), is conceived of as an important mechanism for stem cell pluripotency and aging (Fig. 7). Adult stem cells would be a good model system to study how cellular senescence is occurred. Using adult stem cell aging model, one might be able to detect or examine specific chemicals or toxicants during cellular senescence. Finally, the fact is that adult human stem cells exist in most, if not all, human organs. These cells appear to be ‘‘committed’’ to give rise to the cell types of the organ from which they were isolated, having organ-specific markers, as well as having the

Oct4 gene expressed. For example, the human breast epithelial cell expresses the estrogen receptor (Kang et al., 1997), where the human liver expresses albumin (Kim et al., 2009). That these adult stem cells are indeed capable of producing both a 3D structure similar to that found in vivo, one needs only to view the structure formed when adult human, estrogen receptor– positive, connexin43-negative cells, and Oct4A-positive cells are plated on Matrigel (Fig. 8). This raises an important issue for considering the use of human stem cells for toxicity testing. Currently, for example, 3D and tissue-like in vitro systems for various tissues are being generating, for example, hepatocytes, lens, or skin. However, if one is screening for carcinogens, assuming that the adult stem cell is the target cell for initiating the carcinogenic process (Trosko, 2003b; Trosko et al., 2004), one should use the adult stem cell as the target (Trosko, 2008a, 2008b), not the differentiated somatic cell, such as a mature polyploid hepatocytes that might be derived from either an embryonic stem cell or even a mesenchymal stem cell. The hepatocytes of the liver, themselves, represent another complex mixture (Duncan, et al., 2010). These pure differentiated hepatocytes could be used to screen for hepatocytotoxicants, such as chloroform. Second, if the stem cell possesses expressed drug

FIG. 7. Schematic diagram of senescence regulation in young MSCs and HDAC inhibitor-mediated senescent MSCs. (a) In young MSCs, RB is hyperphosphorylated and expression of EZH2, SUZ12, and c-MYC is controlled by free E2F bound to their promoter region. HDAC inhibitors dephosphorylate RB to bind to E2F transcription factor to repress its transcriptional activity and silence EZH2, SUZ12, and c-MYC genes. Because c-MYC is upstream regulator of BMI1, HDAC inhibition results in BMI1 downregulation. (b) p16INK4A is repressed in young MSCs because of PcG-induced H3K27Me3 enrichment. Repression of PcG expression level and increase of JMJD3 expression level caused by HDAC inhibition leads to demethylation of H3K27 and p16INK4A expression to senesce MSCs (Jung et al., 2010).

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FIG. 8. Human breast epithelial cell (HBEC) colonies on plastic and organoids on Matrigel formed from two types of normal HBEC’s. type 1 and type II colonies developed on plastic (A and C, respectively) are morphologically distinguishable. On Matigel, type II cells typically formed spherical organoid (s.o.) (D), whereas type I cells formed a limited number of bud-like (B) structures and acini (data not shown). The combination of type I and type II cells (E; two types of cells on plastics) in 1:2 or 1:3 ratios can generate many budding (B) ductal (D) structures in Matrigel (F) in 2–3 weeks. Credit to http://cancerres.aacrjournals.org/misc/ifora.shtml.

transporter genes (Kim et al., 2002), then using potential toxic chemicals might be pumped out of these stem cells. Therefore, one might get negative results looking for mutations in the stem cells. However, any chemical, especially at noncytotoxic concentrations, could induce intracellular signaling, which, in turn, could alter the stem cell fate (to proliferate, differentiate, apoptose, or senesce). Any agent that could cause an adult stem cell to have these phenotypic-altering abilities could have either beneficial or toxic side effects. One needs only to view the effects of thalidomide on being a teratogen in utero (Manson, 1986), while being an potential anti-angiogenesis anticancer drug (D’Amato et al., 1994), at the same time as being a tranquilizer (Franks et al., 2004). By using adult stem cells to produce in vivo–like 3D structures, in which all (or most) cell types are present, one might be mimicking real human tissue without having to obtain tissue biopsies every time one wants to screen for new drugs or test the safety of some chemical. Conceivably, by obtaining

human adult stem cells from a finite number of organs (e.g., liver, brain, skin, lung, kidney, breast, pancreas, prostrate) from the female and male, respectively, one could set up a battery to test for some of the major organs that might be subject to toxin/ toxicant-induced diseases. For example, neural stem cells (NSCs) have a capacity for self-renewal and can differentiate into multiple cell types, such as astrocytes, oligodendrocytes, and neurons (Lim et al., 2007). The fate of stem cells is regulated by many factors, such as Wnt/Bmp, that maintain multipotency and suppress differentiation (Votteler et al., 2010). Neurosphere 3D culture methods have been established for the enrichment of NSCs (Fig. 9). During neurosphere culture, drugs or toxicants can be assessed whether some drugs or any chemicals might affect to form neurospheres. In our recent studies, normal neurospheres contain many nestinpositive cells, which is a marker for NSCs. In neurodegenerative diseases, including the Niemann-Pick type C disease (NPC), neurospheres could not be formed (Kim et al., 2008b;

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regenerative therapy but could have many other potential uses for drug screening and toxicity testing. Adult stem cells, those derived from adult organisms, such as adipose-derived mesenchymal stem cells, could be induced to generate many cell types for both regenerative therapy and for drug discovery/ safety assessment. In addition, the organ-derived adult stem cells can be used to develop 3D organoids, mammospheres, pancreaospheres, neurospheres, which could be used to screen for those agents that could alter the symmetric/asymmetric cell divisions during the 3D development, as well as inducing or blocking apoptosis or senescence. Together with co-culture with cells of the immune system, these 3D human tissue–specific organoids, from males and females, might bring toxicity testing closer than what is currently available. However, only with careful and rigorous calibration and controlled conditions will these new options be put into routine usage.

SUMMARY FIG. 9. We combined two images of nestin (green) and iNOS (red) in NSCs from wild-type and NPC1
/
mice. Nestin (green) was weakly expressed, whereas iNOS (red) strongly expressed in NSCs derived from brain (E16) of Niemann-Pick type C disease mice but NSCs derived from brain (E16) of wild-type mice only expressed nestin, not iNOS (Kim, et al., 2008b).

Yang et al., 2006). Therefore, some drug or chemicals can be tested in the recovery of neurosphere-forming ability in neurodegenerative disease. In this study, it was showed that neurosphere-forming ability was recovered after treatment with L-NAME, NO inhibitor in neurospheres derived from NPC mice. This result indicated that neurosphere assay might be good screening tools for the neurodegenerative diseases. By using induced pluripotent stem cells, at this moment, patient-specific iPS cells can be generated but we cannot put back to the patients because of teratoma formation. However, the patients with genetic diseases–specific iPS cells will be very useful for patient-specific drug testing and screening in vitro and for understanding the diseases in near future (Hanna et al., 2007). The embryonic stem cells could be used directly for those toxic agents that could lead to embryo lethality and possibly to some aspects of teratogenesis. However, as in the case of thalidomide-induced teratogenesis, the effect was not on embryonic stem cells but some committed adult stem cell later in embryonic-fetal development. By screening for agents that might affect symmetrical or asymmetrical division of embryonic stem cells could provide valuable insights to the signaling pathways and genes controlling these two choices a stem cell can make. This could be a very important area of toxicology, in that to alter the number of stem cells during embryonic or fetal development might affect any stem cell–based developmental or disease process later in life (the ‘‘Barker hypothesis’’). All induced or iPS cells might have limited use for human

With the general recognition in the field of toxicology that our current concepts, techniques, and strategies for assessing the toxicity of chemicals is simply inadequate for an accurate prediction to the human situation, emerging scientific evidence, including the possibility of using human stem cells, grown in 3D, and of the inclusion of epigenetic endpoints for toxicity, has opened up new potential means to assess potential harm to human health. Because mutating genes in stem cells, altering the numbers (increasing or decreasing) and abnormally modifying the normal expression of genes in stem cells, could affect homeostatic control of tissue development, various stem cell– dependent diseases could result. Understanding the basic biological characteristics of both human embryonic and adult stem cells and the roles they play in normal development, as well as all the complex factors regulating normal quiescence in their niches, in proliferation, in differentiation, in apoptosis, and in senescence, could help to develop in vitro 3D screening assays that could assist in their use to screen for new drugs and assess toxicities of chemicals. Included in the newer strategy to identify how chemicals might affect toxicities, such as birth defects, cancer, immune modulation, chronic inflammation–related diseases, neurological and reproductive dysfunctions, assays to detect modulation of GJIC should be considered. In addition, given the potential of using human stem cells for regenerative medicine or stem cell therapy, understanding the potential toxicities of stem cells should prevent the unknown consequences when placed back into the human recipient. With individual genetic differences, gender factors, developmental stages, unique characteristics of organ-specific stem cell niches, unique repair and drug transporter functions of stem cells, as well as all the other limitations of any in vitro assay (lack of immune functions, diurnal/physiological functions, etc.), the task ahead will not be easy. Yet the potential of obtaining new mechanistic insights to the roles of stem cells in various chemical-induced toxicities,

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such as cancer, in utero effects seen in late life (the Barker hypothesis), and in the aging process, is possible.

Budunova, I. V., and Williams, G. M. (1994). .Cell culture assays for chemicals with tumor promoting or inhibiting activity based on the modulation of intercellular communication. Cell Biol. Toxicol. 10, 71–116.

FUNDING

Buesen, R., Genschow, E., Slawik, B., Visan, A., Spielmann, H., Luch, H., and Seller, A. (2009). Embryonic stem cell test re-mastered: comparison between the validated EST and the new molecular FACS-EST for assessing developmental toxicity in vitro. Toxicol. Sci. 108, 389–400.

A Seoul National University ‘‘World Class University Invited Professor’’ (to J.E.T., K-S.K.); The Korea Government (National Research Foundation of Korea grant, MEST, 2010-002065).

ACKNOWLEDGMENTS

We thank Ms Olga Olowolafe for her excellent secretarial assistance and Mr Hyung-Sik Kim for his efforts in designing the figures. In addition, authors want to thank Dr Parmender Mehta for allowing us to use his figure 2.

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