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1 Introduction

Humans are exposed to a variety of xenobiotic compounds in-cluding pharmaceuticals, chemical substances, cosmetics, food additives, and plant protection products. Before these products can be placed on the eU market, their potential adverse effects in humans need to be tested. For all these substances, reliable data guaranteeing their safety for man and his environment are required. Current pre-clinical test systems often are based on animals or animal-derived cells, which may make them unrep-resentative of the human situation, due to inter-species differ-ences (Nau, 1993; tzimas et al., 1994; Miller and Strömland, 2011). Another drawback of the current test systems is the relatively high number of laboratory animals required for toxi-cological research and regulatory testing. Under the ReACH (Registration, evaluation, Authorisation and restriction of Chemicals) legislation, more than 30,000 chemical substances need to be tested requiring up to 10 million experimental ani-mals in the years ahead (Gilbert, 2010; european Commission, 2006). In recent years, substantial scientific and financial ef-forts have been made toward the development of alternative methods to toxicity studies using laboratory animals. In that context, in vitro models based on human target cells became attractive, in particular hepatocytes isolated from human liver. Drawbacks, however, include the limited tissue availability due to intensive transplantation programs and the fact that iso-lation often yields a low cell number and variable reproducibil-ity due to inconsistent human donor material and phenotypic instability over time (Guillouzo, 1998; Guguen-Guillouzo et al., 2010). An alternative approach is the directed differentia-tion and/or reprogramming of human pluri- and multipotent

stem cells into the desired cell type. this review discusses the unique properties of different types of stem cells, the differ-ent strategies used to generate human target cells, and their potential applications in drug efficacy testing and toxicologi-cal investigations. An overall scheme, illustrating these events, can be found in Figure 1.

2 Classification and properties of human stem cells

2.1 Human embryonic stem cells (hESCs)Stem cells are a unique source of self-renewing cells within the human body. the totipotent fertilized egg represents the ultimate stem cell that gives rise to all embryonic and extra-embryonic structures of the developing embryo (Jung, 2009). heSCs, derived from the inner cell mass of the blastocyst, possess the ability to self-renew over a long period (>500 doubling times) without undergoing senescence (Wobus and Boheler, 2005; Avery et al., 2006). Usually, telomere short-ening is the main actor of “replicative senescence,” but as heSCs express high levels of telomerase activity, they can replace their telomere ends, delay cell senescence, and be cultured in vitro as permanent cell lines without the need for immortalization (Zeng and Rao, 2007). Another key property of heSCs is their ability to differentiate into all cell types of the human body, making them so-called pluripotent stem cells. they generate all cell types of the three embryonic germ layers, including functional ectodermal, mesodermal, and endodermal progeny. Key regulators are octamer binding transcription factor 4 (OCT4), SRY (sex determining region Y)-box 2 (SOX2) and NANOG (Avery et al., 2006; Niwa,

Focus on Stem Cells as Sources of Human Target Cells for In Vitro Research and Testing Joery De Kock, Robim M. Rodrigues, Jennifer Bolleyn, Tamara Vanhaecke, and Vera RogiersDepartment of toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel (VUB), Brussels, Belgium

SummaryThe development and use of new chemical and biological entities require reliable data on their potential adverse effects in humans. Current test systems often are based on animals or animal-derived cells and are not fully representative of the human situation. Therefore, new and optimized human-specific test systems that generate human relevant data are urgently needed. Since the knowledge with respect to stem cell technology has been growing steadily, human stem cell-based in vitro models have become an attractive alternative. Moreover, human stem cell-derived target cells encourage the introduction of functional in vitro models, relevant for the human situation. They can be applied in research and in regulatory testing. In this review we will discuss the unique properties of different types of stem cells, the diverse strategies used to generate human target cells from stem cells, and their potential applications in drug efficacy and toxicology testing.

Keywords: directed differentiation, somatic reprogramming, stem cells, human target cells, pluripotency, drug discovery, toxicological testing

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2007). Moreover, thousands of individual cell lines are avail-able, established from heSCs from surplus embryos after in vitro fertilization (löser et al., 2010).

2.2 Human induced pluripotent stem cells (hiPSs)takahashi and Yamanaka developed a break-through tech-nique in stem cell research by describing the reprogramming of human somatic fibroblasts into primitive pluripotent stem

cells by over-expressing OCT4, SOX2, Krüppel-like factor 4 (KLF4) and v-myc myelocytomatosis viral oncogene ho-molog (MYC) (takahashi and Yamanaka, 2006; takahashi et al., 2007). these hiPSs are similar to heSCs in the sense that they also express pluripotency genes, have telomerase activ-ity, and are able to differentiate into all cell types of the three embryonic germ layers (Maherali et al., 2007; Okita et al., 2007; Wernig et al., 2007).

Fig.1: Strategies for generating human target cellsHuman pluri- and multipotent stem cells can be isolated and/or generated from humans at different stages of life. hESCs are isolated from the inner cell mass of the blastocyst. These pluripotent hESCs can differentiate into all somatic cell types of the three germ layers. Multipotent hASCs are isolated from postnatal human tissue and can differentiate into many different somatic cell types. hiPSs can be generated by reprogramming somatic cells into a pluripotent state. Human target cells can be isolated or obtained by directed differentiation of stem cells using recombinant growth factors, small molecules, cell-cell contacts, and ECM or by direct conversion of somatic cells using viral vectors, miRNAs, modified mRNAs, Cre/LoxP- or piggyBac systems and small molecules. The outcomes of these strategies are customized human target cells that can be used in drug screening, pre-clinical and regulatory testing, and disease modeling. Abbreviations: ECM=extracellular matrix; hASC, human adult stem cell; hESC, human embryonic stem cell; hiPS, human induced pluripotent stem cell; miRNA, microRNA; mRNA, messenger RNAThe figure was produced using Servier Medical Art.

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2.3 Human adult stem cells (hASCs)More than a decade ago, adult stem cells, in contrast to their pluripotent embryonic counterparts, were considered to be lineage-restricted cells, thus incapable of overcoming embry-onic-bound restrictions. In recent years, several publications reported the existence of multipotent postnatal stem cells in bone marrow, skin, neuronal, and adipose tissue, giving rise to various cell types other than their tissue of origin, both in vivo and in vitro (Ferrari et al., 1998; Snykers et al., 2006, 2007; Zech et al., 2007; Jinno et al., 2010; De Kock et al., 2009, 2011a; Al Battah et al., 2011). In contrast to heSCs and hiPSs, multipotent hASCs undergo replicative senescence and, consequently, their lifespan in culture is limited to about 50 cell divisions. the ongoing quest for easily accessible and readily available sources of multipotent hASCs has provided evidence for the existence of “stem cell stores” in adult tissues, such as human skin and adipose tissue, in addition to the well-known mesenchymal stromal cells from bone marrow (leeb et al., 2011).

3 Strategies to generate human target cells using stem cell technology

to generate the desired human target cell from stem cells, two distinct strategies can be applied, i.e. somatic reprogramming or directed differentiation.

3.1 Somatic reprogramming as a tool to generate human target cellsSince Yamanaka and colleagues introduced iPS technology, reprogramming or conversion of one somatic cell type into another cell type, either pluripotent or somatic, has become common practice. the concept that transcription factors are key mediators of cellular identity has gained worldwide ac-ceptance and forms the basis of modern iPS technology. the first hiPSs were generated using retroviral vectors encoding for OCT4, SOX2, KLF4, and MYC, allowing prolonged trans-gene expression (takahashi and Yamanaka, 2006; takahashi et al., 2007). Retroviral reprogramming, however, requires a high viral titer and multiple integrations of the reprogramming genes, resulting in an increased risk of oncogenic events and other ge-nomic and epigenomic abnormalities (Okita et al., 2007; Gore et al., 2011; Hussein et al., 2011; lister et al., 2011). Another drawback of retroviral reprogramming is the necessity for cell division, making slow proliferative cells difficult to reprogram. lentiviral vectors, able to transfect both proliferating and non-proliferating cells, can overcome this problem (Patel and Yang, 2010). today, multiple non-viral strategies are also being ex-plored, avoiding genetic alteration of the reprogrammed cells. Both the loxP/Cre recombinase (Chang et al., 2009) and the piggyBac transposon systems (Woltjen et al., 2009, 2011) have been used effectively to deliver various reprogramming fac-tors. An advantage of such systems is the option to remove the exogenous transgenes after successful reprogramming (Chang et al., 2009; Kaji et al., 2009; Woltjen et al., 2009, 2011). Re-

cently, synthetic modified mRNAs encoding for reprogram-ming factors have been developed to successfully reprogram somatic cells into a pluripotent state. the transient nature of the expressed proteins, following mRNA transfection, minimizes the risk of oncogenic events (Warren et al., 2010). Recently, microRNAs (miRNAs) were found to play a major role during embryonic development. those small (19-25 nt), endogenous encoding RNAs also have been efficiently used to enhance the reprogramming of somatic cells into a pluripotent state, either in conjunction with or in the absence of other reprogram-ming factors (Mallanna and Rizzino, 2010; Sun et al., 2010; Anokye-Danso et al., 2011; Bolleyn et al., 2011; Onder and Daley, 2011). In addition, several low molecular weight organ-ic compounds, called “small molecules,” have been identified that are able to increase the efficiency of reprogramming or substitute one or more reprogramming factors. Several of these are epigenetic modifiers such as histone deacetylase inhibitors or DNA-methyltransferase inhibitors (Feng et al., 2009; efe and Ding, 2011). Increasing evidence suggests that epigenetic regulation is mandatory to the preservation, acquisition, or loss of the primitive stem cell state. therefore, since pluripotent stem cells harbor a hyperdynamic and more open chromatin structure compared to their differentiated progeny, remodeling of the epigenome appears to be a crucial barrier that needs to be addressed in somatic cell reprogramming (Meshorer et al., 2006; Meissner et al., 2007; Sridharan et al., 2009). It appears that the epigenetic memory of the donor tissue of origin is re-tained by reprogrammed somatic cells, favoring differentia-tion along lineages related to the donor cell type (Chin et al., 2010; Kim et al., 2010; Polo et al., 2010; Bar-Nur et al., 2011). these advances in somatic reprogramming have not only con-tributed to a more efficient generation of induced pluripotent stem cells but also to successful conversion of somatic cells into other functional cell types of interest, as such, bypassing a pluripotent state. During the past year, several high impact publications report the successful reprogramming of somatic fibroblasts into in vitro and in vivo functional neuron-like, car-diomyocyte-like and hepatocyte-like cells by overexpression of key lineage specific transcription factors (Efe et al., 2011; Huang et al., 2011; Pfisterer et al., 2011; Sekiya and Suzuki, 2011; Son et al., 2011).

3.2 Directed differentiation using nature as a field guide to generate human target cellsDuring the embryonic development in vivo, the micro-environ-ment of developing cells is continuously changing, following successive biological events that result in the specification, dif-ferentiation, and maturation of primitive stem cells into spe-cialized somatic cells. each step of this developmental process is tightly regulated by intra- and extracellular communication, as well as by cell autonomous mechanisms. Based upon the knowledge of how a developing embryo produces a particular cell type, our laboratory developed a successful in vitro dif-ferentiation strategy using normal biological development as a field guide (Snykers et al., 2009). Other authors also applied this strategy to other cell types (Germain et al., 2010). A com-

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4.1 Genotoxicity/mutagenicityFor instance, in vitro models based on pluripotent stem cells are useful to analyze genotoxic and mutagenic effects of drugs and xenobiotics (reviewed by Wobus and löser, 2011). they reduce, but do not replace, time-consuming and expensive in vivo germ cell tests, including mammalian spermatogonial assays, chromosome aberration tests, and dominant lethal (Russell and Russell, 1954; Ehling, 1974), specific locus (Eh-ling et al., 1978; Russell et al., 1981), and mouse heritable translocation assays (eastmond et al., 2009).

4.2 Developmental toxicityStem cell-based in vitro models also could be useful in pre-dicting human-specific developmental toxicity. Recent proof-of-concept studies indicated that results obtained using stem cell-based models can, to a certain degree, correlate with known embryotoxic effects caused by a number of drugs or chemicals (reviewed by van Dartel and Piersma, 2011). Some key issues, however, need to be addressed before these assays can be implemented into routine procedures for developmental toxicity testing, replacing the time-consuming and expensive in vivo tests that require high numbers of laboratory animals. these include the establishment of reliable and reproducible differentiation strategies and determination of the predictivity, sensitivity, and specificity of the respective test system for a wider panel of drugs and chemicals (reviewed by van Dartel and Piersma, 2011).

4.3 Organ-specific toxicityHuman target cells derived from pluri- or multipotent stem cells are also a potentially interesting cell source to evaluate organ-specific toxicity, including liver, heart, and the nervous system.– Liver toxicity: Functional hepatocyte-like cells can be gen-

erated from stem cells upon sequential exposure to hepa-togenic growth factors and cytokines (reviewed by Snykers et al., 2009) or direct somatic reprogramming (Huang et al., 2011; Sekiya and Suzuki, 2011). Obtaining mature hepato-cytes from stem cells remains a major challenge. Critical steps, such as reproducibility, genetic and epigenetic sta-bility, and up-scaling still must be overcome before large numbers of metabolically competent hepatocytes can be generated and used in toxicological testing. today, an often used surrogate for human primary is HepaRG cells. this is a liver cell line derived from a human liver carcinoma (reviewed by Guguen-Guillouzo et al., 2010).

– Cardiotoxicity: One of the leading reasons for drug attri-tion, other than hepatotoxicity, is cardiotoxicity (Guo et al., 2011). It is therefore critical to screen for factors that may induce or enhance cardiac failure during the development of new pharmaceuticals. Within this field, stem cells have emerged as a promising human alternative to reduce, re-place, and/or refine present animal models. Currently, the strategy is well established for the in vitro development of

mon method for controlling cell fate in vitro is the addition to the differentiation media of recombinant growth factors that play a well-known role in the differentiation of the desired cell type in vivo. this strategy has been especially success-ful in mimicking the earliest steps of embryonic development, resulting in commitment to one of the three germ layers: ec-toderm, mesoderm, and endoderm (Perrier et al., 2004; De Kock et al., 2009; Oldershaw et al., 2010). Besides the suc-cessful application of recombinant growth factors in directed differentiation, additional strategies (e.g. small molecules, xenogenic proteins, cell culture conditions...) are required to produce fully functional human target cells. Small molecules can offer a valuable alternative and/or supplement to the es-tablished approaches with recombinant growth factors. More specifically, addition of small molecules can induce, facilitate, or inhibit signaling through a specific developmental pathway, resulting in the desired specification of the target cell (Frank-Kamenetsky et al., 2002; li et al., 2008; efe and Ding, 2011). In comparison to xenogenic proteins, small molecules are less expensive, non-immunogenic, more stable, and have less batch-to-batch variability. Apart from controlling cell fate by soluble medium factors, reconstruction of in vivo-like cell-cell and cell-matrix interactions can also enhance directed differ-entiation of stem cells into the desired human target cell. Co-culture systems, in which differentiating cells are plated in the presence of supporting cells, can provide the differentiating stem cells with the appropriate micro-environment, including intercellular communication and/or the secretion of a mixture of growth factors and other signaling molecules, to guide their differentiation (Mummery et al., 2003). Furthermore, extracel-lular matrix (eCM) proteins can complete the reconstitution of the in vivo micro-environment, providing a natural backbone and additional growth factors necessary for differentiation (De Kock et al., 2011b; Kim et al., 2011).

4 Stem cells and their progeny in drug screening and toxicological testing

Because of species differences in cellular metabolism, toxic events occurring in man frequently are not picked up in cells of experimental animals. Consequently, in order to obtain rel-evant data with respect to potential adverse effects of substanc-es, human cells should be used. In this regard, the availability of pluri- and multipotent stem cells may play a key role. these primitive cells can be differentiated or reprogrammed into functional somatic progeny, exhibiting many of the properties of the human target cells of interest and bypassing the limita-tions of currently used cell lines and primary cells (Biernaskie et al., 2007; lavoie et al., 2009; efe et al., 2011; Huang et al., 2011; Pfisterer et al., 2011; Sekiya and Suzuki, 2011; Son et al., 2011; Sullivan et al., 2010). Potential applications have already been proposed in various fields. A number of these are mentioned below.

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man-relevant toxic response. As the in vivo mechanisms driv-ing cell differentiation and dedifferentiation are still not fully understood, it is not yet feasible to pursue in vitro tests that predict entirely the in vivo situation. However, it is strongly believed that once the in vivo processes are sufficiently under-stood, a physiologically relevant in vitro system can be estab-lished. Human stem cell-based in vitro tests, relying on human biological systems, can be useful for toxicological research and would be more relevant than the traditionally used ani-mal-based models. Although the use of stem cells holds great promise, enormous challenges are still ahead. It is critical to address the poor reliability of stem cell-based in vitro tests due to the absence of method harmonization and standardization. An issue often faced while performing stem cell-based tests is intra- and inter-laboratory variability. Proper characteriza-tion of the starting material, for example by the introduction of well-defined molecular signature profiles of the used stem cell populations, and standardization of differentiation and/or reprogramming protocols can help to solve this problem. Another challenge ahead is the insufficient knowledge with re-spect to pathways and key events of toxicity within organisms and biological systems in health and disease. Customized hu-man target cells and “disease-in-a-dish” models derived from stem cells can help to clarify these in vivo processes and con-tribute to a targeted approach for identifying key pathways and networks in vitro.

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calcium handling mechanisms, ion channels, and regulatory proteins important for a mature repolarization phenotype of cardiomyocyte-like cells. Despite these advances, several aspects, including cell purity and strategies to provide the correct micro-environment to enhance further maturation of the stem cell-derived cardiomyocytes, still need to be ad-dressed (reviewed by Mandenius et al., 2011).

– Neurotoxicity: the use of stem cells in neurotoxicology and developmental neurotoxicity is another emerging field. Neural progenitor cells, either isolated from neuronal tissue or derived from pluri- or multipotent stem cells, can give rise to a mixed population of neurons and glial cells that can be used to evaluate neurotoxicity in vitro. A relatively sensi-tive model in developmental neurotoxicity is neurite out-growth. In most of these studies stem cells are differentiated in the presence of the chemical of interest, and the resulting populations of neurons and/or glial cells are compared to the control conditions (Radio and Mundy, 2008; reviewed by Breier et al., 2010).

4.4 Disease-specific toxicityAnother application of stem cells and/or their progeny is to screen for novel drugs and adverse effects in so-called “disease-in-a-dish” models. these models can be developed from human stem cells isolated and/or generated from disease-specific patients. they offer human information additional to existing knowledge obtained through whole animal and/or animal cell-based systems (reviewed by Saha and Hurlbut, 2011). As heSCs cannot be de-rived from adult individuals, their application in the field of in vitro disease modeling remains limited. In contrast, as hiPSs can be derived from virtually any individual, the generation of dis-ease-specific hiPS cell lines permits the development of so-called “disease-in-a-dish” models (Park et al., 2008; Carvajal-Vergara et al., 2010). therefore, hiPSs represent a promising tool for the near future to study human disease phenotypes at the cellular and molecular level. hiPS derived cell lines may produce more reliable experimental human disease models than non-human animal models. They can provide a virtually infinite supply of cells without requiring additional tissue donation. However, as hiPSs undergo reprogramming, recent genomic and epigenomic analyses revealed a number of abnormalities that could affect and limit their further use (Gore et al., 2011; Hussein et al., 2011; lister et al., 2011; Pera, 2011). However, other interesting stem cell sources for disease modeling are also available, including hASCs. they can be derived from virtually any individual and produced in large quantities. In contrast to hiPSs, they do not require cellular reprogramming. As a consequence, they harbor a larger (epi)genomic stability but their shorter lifespan and dif-ferentiation capacity, to a certain extent, limit some applications (Mimeault and Batra, 2009).

5 Future perspectives

the ultimate cell system for drug screening and toxicity testing should express in vitro what is required in vivo to produce a hu-

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Correspondence toJoery De KockDept. toxicologyCenter for Pharmaceutical ResearchVrije Universiteit Brussel (VUB)laarbeeklaan 1031090 BrusselsBelgiumPhone: +32 2 477 4517Fax: +32 2 477 4582e-mail: jdekock@vub.ac.be

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