Human embryonic stem cells: Current technologies and emerging industrial applications

Transcription

1 Critical Reviews in Oncology/Hematology 65 (2008) Human embryonic stem cells: Current technologies and emerging industrial applications Caroline Améen a, Raimund Strehl a, Petter Björquist a, Anders Lindahl b, Johan Hyllner a, Peter Sartipy a, a Cellartis AB, Arvid Wallgrens Backe 20, Göteborg, Sweden 1 b Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska University Hospital, Göteborg, Sweden Accepted 27 June 2007 Contents 1. Introduction Pluripotent hes cells Establishment of hes cell lines Cultivation of hes cell lines Spontaneous differentiation of hes cells Characterization and quality control of hes cell lines Xenofree derivation and cultivation of hes cells Derivation of functional cells from hes cells Embryogenesis Hepatocytes Hepatogenesis Stem cell differentiation to hepatic cells Differentiation of hes cells to hepatic cells Cardiomyocytes Cardiogenesis ES cell differentiation to cardiac myocytes Characteristics of hes-cm Cell selection and enrichment strategies Cardiac progenitor cells Applications of hes cells Human ES cells in regenerative medicine The heart as a regenerative organ Human ES cells and cell therapy for cardiac regeneration Concerns with hes cells in cell therapy Activation of endogenous stem cells Human ES cells for use in drug discovery and toxicity testing Developmental toxicology Applications for hepatocytes derived from hes cells Applications for cardiomyocytes derived from hes cells Conclusion Reviewers Corresponding author. Tel.: ; fax: address: peter.sartipy@cellartis.com (P. Sartipy) /$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi: /j.critrevonc

2 C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) Conflicts of interest Acknowledgements References Biography Abstract The efficiency and accuracy of the drug development process is severely restricted by the lack of functional human cell systems. However, the successful derivation of pluripotent human embryonic stem (hes) cell lines in the late 1990s is expected to revolutionize biomedical research in many areas. Due to their growth capacity and unique developmental potential to differentiate into almost any cell type of the human body, hes cells have opened novel avenues both in basic and applied research as well as for therapeutic applications. In this review we describe, from an industrial perspective, the basic science that underlies the hes cell technology and discuss the current and future prospects for hes cells in novel and improved stem cell based applications for drug discovery, toxicity testing as well as regenerative medicine Elsevier Ireland Ltd. All rights reserved. Keywords: Human embryonic stem cells; Differentiation; Cardiomyocytes; Hepatocytes; Drug discovery; Toxicology; Regenerative medicine 1. Introduction Pluripotent human embryonic stem (hes) cell lines were successfully derived from the inner cell mass (ICM) of human blastocysts in the late 1990s [1]. With their unparalleled growth capacity and unique developmental potential to differentiate into almost any cell type of the human body, these cells are expected to revolutionize biomedical research worldwide. The hes cell technology has the potential to open novel avenues both in basic and applied research as well as in therapeutic applications. In many biomedical disciplines, such as drug discovery and toxicology studies, the lack of functional human cell systems makes the research process inefficient and the outcome sometimes inaccurate. Based on both clinical and financial grounds, it is thus crucial to develop innovative tools that increase the speed of drug development in a cost-efficient and clinically relevant manner. The access to undifferentiated hes cell lines and derivatives thereof offers great opportunities in a wide range of applications, spanning from early target identification and validation studies, via cellular screening and lead optimization, to the use of functional human cells in toxicity assessment and safety pharmacology as well as in various disease models (Fig. 1). There is also much optimism concerning the use of selectively differentiated pluripotent hes cells in cell therapy, initially targeting degenerative diseases. In essence, many of the shortcomings in the drug development process today could conceivably be overcome or improved by exploitation of hes cell technology, optimally promoting the discovery of new drugs and treatments. In this feature, we review the basic science that underlies this exciting field by describing the establishment and maintanence of pluripotent hes cells along with the directed differentiation of these cells into functional cell types. We further discuss the current and future prospects for hes cells in some novel and improved stem cell based applications for drug discovery, toxicity testing as well as regenerative medicine. The focus throughout this review is on hes cellderived cardiomyocytes and hepatocytes, since these two cell types are central in the drug development process. The derivation and application of other cell types are omitted due to space limitations and are reviewed elsewhere. 2. Pluripotent hes cells 2.1. Establishment of hes cell lines There are a number of sources of human stem cells with varying degrees of developmental potency. Multipo- Fig. 1. Potential use of hes cells in drug discovery. The use of hes cells and their specialized progenies in drug discovery spans from early target identification and validation studies, via the use of functional human cells in screening and metabolism studies, to the use of various stem cell technologies in toxicity testing. In addition, an interesting and prodigious future opportunity is to develop drugs that affect endogenous pools of stem cells to repair local defects in the human body.

3 56 C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) tent adult stem cells can be derived from the bone marrow or from specific organs, while different types of multipotent fetal stem cells also can be obtained from umbilical cord blood or from aborted human fetuses [2]. The method for the derivation of hes cell lines [1] was initially adapted from the previously developed methods for mouse and primate ES cells. To establish a new hes cell line from the ICM of a blastocyst, the expanded blastocyst is first incubated with pronase to digest the surrounding zona pellucida (ZP). Subsequently the ZP-free blastocyst is treated with anti-human whole serum antibody and guinea pig complement. This process, which is termed immunosurgery, lyses the trophectoderm by an antibody/complement reaction. The isolated ICM is then placed on a layer of mitotically inactivated mouse embryonic fibroblasts (MEF) feeder cells in a gelatin-coated tissue culture dish. The initial ICM outgrowth is usually dissected and transferred to new culture dishes after 7 14 days. Successful propagation of the ICM is associated with the appearance of cells with undifferentiated hes cell morphology, whereas contaminating cell types such as primitive endoderm and trophectoderm disappear [3]. The culture medium used for hes cell cultivation is based on Dulbecco s modified Eagle s medium supplemented with 20% Knockout serum replacement or more historically 20% fetal bovine serum. To date, hes lines have been derived in a number of independent laboratories worldwide using the traditional derivation method [4 6] or alternative approaches in which immunosurgery is not performed, such as whole embryo culture or partial embryo culture [7,8]. The hes cell lines can be maintained in culture indefinitely and exhibit a stable developmental potential to differentiate into all the cells of the human body. Unlike mouse ES cells, hes cells can also give rise to trophectoderm-like cells in vitro [9]. The procurement of hes cell lines has been surrounded by ethical and legal considerations which, in most parts of the world, have led to the establishment of guidelines and regulations concerning stem cell research Cultivation of hes cell lines The proper maintenance and expansion of hes cells is one of the most important issues in the study of hes cells as this procedure generates the starting material for all subsequent steps and therefore determines the baseline quality. In order to expand hes cell lines in an undifferentiated and pluripotent state, the hes colonies are traditionally cultivated on a mitotically inactivated MEF feeder layer (Fig. 2a). The feeder layer provides certain currently unknown factors, which support undifferentiated growth of hes cells [10]. Unlike in mouse ES Fig. 2. Morphology of hes cells in different culture systems. (a) hes cell colonies grown on MEF feeder layer. Scale bar = 100 m, (b) hes cell colonies grown in feeder-free culture on Matrigel TM. Scale bar = 250 m, (c) hes cell colonies grown on human feeders. Scale bar = 100 m, and (d) EBs from hes cells in suspension culture. Scale bar = 500 m.

4 C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) cell cultivation, where addition of LIF to the culture medium is sufficient to maintain the cells in an undifferentiated state, human LIF does not prevent hes cells from differentiating [1,5]. The most widely used method to maintain normal hes cells of high quality is propagation using manual microdissection [1,3,5]. In this delicate process, micropipettes or finely drawn Pasteur pipettes are used to dissect individual hes cell colonies into small pieces and subsequently transfer them to fresh culture dishes. This procedure, which requires particularly refined skills, is usually repeated every 4 5 days. The main advantages of the mechanical transfer method lie in the absence of cell-dissociating enzymes and the ability to perform a positive selection at every passage by isolating undifferentiated hes cells from more differentiated cells. However, this method is laborious and time consuming, making it very difficult to process many cells simultaneously. Due to the disadvantages inherent to the traditional culture protocols for hes cells, numerous attempts have been made to develop alternatives which do not require feeder layer preparation or manual passaging. A widely used feeder-free culture system takes advantage of the fact that soluble factors which are necessary for maintenance of undifferentiated hes cells are secreted into the culture medium by the feeder cells. The hes cells therefore can be grown in the absence of feeder cells on a growth substrate such as Matrigel TM using a MEF conditioned medium [11 14] (Fig. 2b). Conditioned medium is usually prepared by overnight incubation with a confluent MEF feeder layer and by subsequent filtration prior to its use for hes cell culture. Other reports indicate that culture additives which activate the canonical Wnt pathway [15], a combination of growth factors such as LIF, transforming growth factor- 1 (TGF- 1) and basic fibroblast growth factor (bfgf) [16], a combination of noggin and bfgf [17] or high levels of bfgf alone [18] may be sufficient to sustain undifferentiated hes cells in the absence of feeders. The use of enzymes for cell dissociation during passage is obviously considerably faster and simpler than microdissection. Therefore, different enzymes such as collagenase IV [14,19], trypsin [20] and dispase [7,21,22] have been employed for the expansion of hes cells. During passage, the hes cell colonies are incubated with enzyme until a suspension of the desired cluster size has been achieved. The suspension is then transferred to new culture dishes. According to several reports, the use of enzymes for hes cell transfer may increase the risk of introducing genomic aberrations during propagation in vitro [23 26]. It is still not clear, however, in which way culture conditions and the occurrence of chromosomal abnormalities relate to each other. Several different cryopreservation protocols have been used for long-term storage of hes cells. The most common method for traditionally cultured hes cells is vitrification of microdissected colonies in open or closed straws [3,27,28]. Enzymatically passaged hes cells have also been cryopreserved successfully by slow freezing of small clusters in cryotubes [13,14,29]. In addition, protocols for the cryopreservation of adherent hes cell colonies have been suggested [30]. The demand for undifferentiated hes cells as well as their differentiated progenies for research and regenerative medicine is expected to increase massively. The culture methods for pluripotent hes cells available today do not allow for the production of large enough cell quantities to meet this demand. In addition, the protocols for the transformation of undifferentiated hes cells to specific functionally differentiated cells are rather unefficient, thus large amounts of the undifferentiated hes cell starting material are required. Considerable effort is now put into the industrial scale-up of hes cell production. Even though attempts have been made to partly automate the traditional manual propagation of hes cells [31], large scale production of hes cells requires new culture technologies which are more robust, efficient and certainly more cost-effective. More efficient feeder systems, such as different types of human feeders [21] or immortalized mouse feeders [32], have been developed and tested for replacement of the laborious and inconsistent preparation of traditional MEF feeders. The use of enzymes instead of microsurgery for passage is an essential prerequisite for the cost-effective production of hes cells [33]. In addition to the above mentioned collagenase IV and dispase, we have been using the recombinant enzyme TrypLE select to passage hes cells maintained on highly supportive human foreskin fibroblast (hff) feeder layers in order to robustly increase the production of undifferentiated hes cells (Fig. 2c). Such an enzymatic culture method fulfills the requirements for future automation using cell culture robots to allow further industrial scale-up [34]. Mouse ES cells have successfully been cultured in stirred bioreactors [35] as well as in perfused reactors [36]. Likewise, bioreactor technology should have great potential for hes cell expansion. Continuous medium renewal in cultures has been found to be beneficial for the maintenance of undifferentiated hes cells as well [37] and different types of rotating microgravity reactors [38] or stirred vessels [39] have been employed for cultivation of differentiating hes cells. Therefore, the use of more advanced perfused reactors or hollow fiber systems for the production of undifferentiated hes cells appears plausible for future development. Scale-up of hes cell cultures will remain difficult though, until the factors that regulate hes cell pluripotency and the maintenance of the undifferentiated state are discovered and fully understood Spontaneous differentiation of hes cells In contrast to embryonal carcinoma cells [40], undifferentiated hes cells have an enormous potential to spontaneously differentiate into various cell types in vitro. Little is known to date about the mechanisms which can trigger the spontaneous differentiation of pluripotent hes cells into a seemingly chaotic mix of differentiated cell types. Unwanted spontaneous differentiation of hes cells is observed to a varying degree under all routine culture conditions and still presents

5 58 C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) one of the major obstacles in hes cell cultivation today. The unique spontaneous differentiation potential of hes cells can on the other hand be harnessed to recapitulate certain aspects of early human embryonic development in vitro and to easily generate a variety of hes cell progenies. Simple spontaneous differentiation of hes cells can be initiated experimentally by discontinuing to passage the hes cell colonies or by withdrawal of conditioned medium. More complex spontaneous differentiation can be obtained by cultivation of the hes cells as three-dimensional aggregates, so called embryoid bodies (EBs) [41]. To initiate the formation of EBs, the hes cell colonies are removed from the supporting feeder layer and allowed to aggregate into spheres. The spheres are usually maintained in suspension culture and give rise to differentiated cell phenotypes of all three germ layers, which arise by a complex pattern of cross-induction (Fig. 2d). Further differentiation can be obtained by subsequently plating the whole EBs or cells released from the EBs onto specific extracellular matrix components Characterization and quality control of hes cell lines Human ES cells can be maintained in vitro indefinitely. The cell lines can be banked and serve as a reproducible, welldefined source for the generation of a large variety of human cells for various in vitro applications as well as for future therapeutic purposes. As hes cells can change in culture due to differentiation or due to genomic alterations, however, it is essential to maintain rigorous quality management and a high level of quality control. Principles well known today in the industrial production of other mammalian cells, such as master cell banks, working cell banks and controlled batches, must be applied to the establishment, banking and production of hes cell lines as well. A number of typical hes cell attributes can be used for their characterization and quality control. Pluripotent hes cells can be characterized phenotypically by their morphology and by their marker expression profile. Undifferentiated hes cells grow as colonies with distinct borders. The hes cells are small, densely packed and exhibit a typical cell morphology with a high nucleus to cytoplasma ratio and large nucleoli. The morphology of spontaneously differentiating hes cell colonies is clearly different and can take on many various forms. The colonies may appear to lose their tight border and cells within the colony may either begin to enlarge, flatten and separate or may pile up and appear thick and opaque. Characteristic cell surface markers of undifferentiated hes cells are the stage-specific embryonic antigen 3 (SSEA-3) [42] and SSEA-4 [43], the high molecular weight glycoproteins tumor rejection antigen 1 60 (TRA-1 60) and TRA-1 81 [44], and the germ cell tumor monoclonal-2 (GCTM-2) antigen [45]. These markers are downregulated upon differentiation. Unlike mouse ES cells, undifferentiated hes cells do not express SSEA-1 [46]. Undifferentiated hes cells display alkaline phosphatase- and telomerase activity [1]. Furthermore, the human POU-domain transcription factor Octamer-4 (OCT-4) [47], Nanog [48] and Sox2 [49] are highly expressed in the undifferentiated state and can be used to monitor the differentiation status of hes cells. For the purpose of quality controlling hes cell cultures, combinations of markers can be measured on the protein level using immunocytochemistry [50], fluorescence activated cell sorting (FACS) [51] or on the gene expression level using real-time quantitative PCR [52,53]. A much discussed aspect of hes cells is their genetic stability in culture [54,55]. Therefore cytogenetic evaluation must be an important element in the quality control of hes cell lines. Examples of the most commonly used methods for cytogenetic analysis of hes cells today are karyotyping, fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) [56,57]. Karyotypes are prepared for analysis by staining the chromosomes to reveal their banding pattern and arranging them as an ideogram. Such karyotypes are used to study gross chromosomal aberrations and may be used to determine other aspects of a hes cell line s genotype, such as sex. FISH uses fluorescent DNA probes that bind to specific parts of a chromosome with which they show a high degree of sequence similarity. Depending on the combination of probes employed, FISH can be used to detect and localize the presence or absence of several specific DNA sequences on chromosomes. CGH allows the analysis of copy number changes (gains or losses) in the DNA content of cells. The method is based on the hybridization of fluorescentlabeled abnormal and normal DNA to metaphase preparations followed by quantitative image analysis of regional differences in the fluorescence ratios. Each of these techniques has its limitations. Traditional G-banding karyotypes are difficult to analyze due to the low resolution banding pattern in stem cells, FISH does not allow screening of all the chromosomes of the genome for chromosomal changes and CGH is unable to detect balanced translocations, mosaicism and ploidy. Therefore the methods are usually used in combination to complement one another. Finer resolution analysis of genomic stability is possible by single nucleotide polymorphism assays, which can also be applied to generate genomic fingerprints of hes cell lines [58]. Another technique with great potential for the molecular cytogenetic characterization of hes cell lines is spectral karyotyping. The technique uses multiple probes to simultaneously visualize all chromosome pairs in different colors [59]. These methods can be used to identify chromosomal aberrations when other techniques are not accurate enough. ES cells have the capacity for extensive self-renewal but possess the ability to differentiate along multiple cell lineages at the same time. Long-lasting changes in gene expression patterns are required during the progression of undifferentiated ES cells towards more differentiated progeny. Such inheritable cellular gene expression memory can be controlled by epigenetic mechanisms, such as DNA methylation or histone modification [60]. Methylation and demethylation of regulatory sequences in the genome are known to

6 have profound effects on cellular fate [61]. Changes in histone modification and DNA methylation may also perturb X chromosome inactivation in hes cell lines, an important mechanism for gene dosage compensation to ensure that female embryos express similar levels of X-linked genes to males [62]. Epigenetic changes during culture may have serious implications for the use of hes cells, especially in regenerative medicine by affecting the differentiation capacity of the cell lines [61]. Increased knowledge of the human epigenome is hence expected to contribute significantly to the future capabilities of hes cell line characterization. The paramount requirement a hes line has to meet is pluripotency, i.e. the hes cell line must have the potential to give rise to derivatives of all embryonic germ layers. Therefore pluripotency testing is an essential part in the characterization and quality control of hes cell lines. Pluripotency can be tested in vitro by letting hes cells spontaneously differentiate via an EB step [3,5]. The differentiated material is then plated into culture dishes and analyzed immunocytochemically for markers specific for the individual embryonic germ layers. Currently, the golden standard for assaying the pluripotency of hes cells is performed by in vivo xenotransplantation of undifferentiated hes cells into severe combined immunodeficiency mice where the xenografted hes cells give rise to teratomas [1,3,5]. These tumors contain various types of tissues representing all three embryonic germlayers (Fig. 3), such as striated muscle, cartilage and bone (mesoderm), gut epithelium (endoderm) and neural rosettes (ectoderm). The tissues show a varying degree of differentiation and can be evaluated histologically, thus providing experimental proof of pluripotency Xenofree derivation and cultivation of hes cells C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) The ultimate potential of hes cells lies in the clinical transplantation of differentiated cells for disorders which arise from loss-of-function of a single cell type such as diabetes or Parkinson s disease. With respect to any future therapeutic application of hes cell derivatives it is important to eliminate the risk of contamination with animal pathogens or immunogenic molecules from the mouse feeder cells or from any animal derived components in the cell culture medium [63]. Therefore any direct or indirect exposure of the hes cell line to animal material has to be avoided during derivation as well as propagation in vitro. One successful approach to avoid exposure of the hes cell line to animal material is to replace the animal derived components in the traditional culture environment with human derived substitutes. The traditionally used mouse feeder cells have been substituted with several different types of feeder cells derived from human tissues [16,64 66] as well as with an autogeneic feeder layer derived from the hes cells [67]. However, the basal medium used in the above studies still contained animal derived proteins. Complete humanization of the feeder layer as well as the culture medium was recently reported by Ellerström et al. [68]. By strict use of only human Fig. 3. Histology of teratoma from hes cells xenografted to SCID mice. (a) Neural rosettes (ectoderm), (b) Hyaline cartilage (mesoderm), and (c) Columnar gut epithelium cells with goblet cells (endoderm). All scale bars = 100 m. or synthetic components during the derivation and propagation process a new xeno-free hes cell line was established. Another promising approach to avoid xeno-exposure of hes cell lines is the use of defined culture environments that are not based on a feeder cell layer or on a feeder cell conditioned medium. Defined feeder-free growth surface

7 60 C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) coatings with individual human extracellular matrix components, such as laminin or fibronectin in combination with non-conditioned serum-free media supplemented with various combinations of human recombinant growth factors, have been employed for the culture [69 71] as well as the derivation of hes cell lines [72]. 3. Derivation of functional cells from hes cells The successful establishment of hes cell lines raises a whole new set of expectations. One big challenge, however, is the directed and controlled derivation of lineage-restricted functional cell types from hes cells. To date, most specialized cells that are derived from hes cells are selected and propagated from an unsorted mix of various spontaneously differentiated cells. It will thus become very important to define the culture conditions and find appropriate isolation methods that will give rise to a high yield of pure populations of differentiated, functional cells intended for future applications both in vitro and in vivo. In this section, we focus on the differentiation of hes cells into hepatocytes and cardiomyocytes as well as their respective characteristic properties. The discussion regarding derivation of other cell types from hes cells is omitted and the interested reader is referred to other excellent reviews on this subject [73 75] Embryogenesis During embryogenesis the distinct cell lineages are established. At the stage of gastrulation, a fundamental step in the development of all types of animals, a gut structure is formed by drawing cells from the exterior to the interior. A three-layered structure is subsequently formed in this process, with the innermost layer of the gut tube forming the endoderm, the outmost layer becomes the ectoderm and the looser tissue between the two forming the mesoderm. These three structures are the germ layers that are common to all higher animals. Together they constitute the first representative organization of the adult body with the gut on the inside, the epidermis on the outside and the loose and hard connective tissue and muscle in between Hepatocytes Hepatogenesis The definitive endoderm, one of the three embryonic lineages, gives rise to the digestive tract and to organs such as pancreas and liver [76]. The induction of hepatic genes requires signaling from at least two different mesodermal tissues; FGFs from the adjacent cardiac mesoderm and bone morphogenetic proteins (BMP) 2 and 4 from the septum transversum mesenchyme [77,78]. These interactions with the endodermal cells are consequently crucial for early liver budding phase. When the hepatic endoderm is specified and the liver bud begins to grow, the cells are referred to as hepatoblasts [79]. These cells seem to be bipotential, capable of differentiating into hepatocytes and cholangiocytes (bile duct cells) [80]. The hepatoblasts associate with endothelial cells to form capillary-like structures [81]. Many signaling molecules and transcription factors are needed for the subsequent growth, maturation and polarization of the liver cells. Although several molecular details have been discovered in rodents, the exact gene regulation in human liver development has not yet been fully characterized [82,83] Stem cell differentiation to hepatic cells The liver is the largest organ in mammals and it serves a variety of important functions. Hepatocytes, the most abundant cells of the liver, perform a number of tasks, including metabolism of most dietary molecules, detoxification of hazardous agents and storage of glycogen. Through the basal membrane, the hepatocyte conditions the venous blood coming into the liver by the secretion of soluble factors. Through its apical membrane, the hepatocyte secretes bile into the canaliculae that join the bile ducts. The majority of the remaining cells of the liver are Kupffer cells, stellate cells, cholangiocytes and various endothelial cells. Altogether, the different cell types are building up the complex threedimensional structure of the human liver. Stem cell differentiation into hepatocytes is of great interest, since an access to large numbers of these cells would enable their use in place of whole organ transplantation as a potential treatment for severe liver diseases. Of specific interest in this review is the idea that a readily source of hepatocytes also substantially could facilitate the development of new drug discovery strategies. Stem cells as promising sources of human hepatocytes in the future can be roughly classified into adult and embryonic stem cells. The adult stem cells could further be divided into intrahepatic and extrahepatic stem cells. The intrahepatic stem cells are referred to as hepatoblasts, hepatic progenitors or liver stem cells, and are a natural source of mature hepatic cells [84 86]. The extrahepatic stem cells can be found in most organs in the human body and some of them have been shown to have the capacity to differentiate in vitro or in vivo to the hepatic lineage [87 90]. This review will focus on the potential of embryonic stem cells to serve as the source for mature hepatic cell types Differentiation of hes cells to hepatic cells For more than 20 years, mouse ES cells have served as a tool for studying the embryonic development. The first report on directed differentiation of ES cells to hepatocytes in vitro was published in 2001 when Hamazaki and colleagues successfully studied hepatic maturation in cultures of mouse ES cells [91]. This report has been followed by numerous studies on mouse ES cell differentiation to the hepatic lineage. Although different culturing techniques have been used, including monolayer cultures [92,93], EB formation [94,95] and co-culturing with liver cells [96], the development of

8 C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) hepatic cells from mouse ES cells was not efficient and was always seen in heterogeneous cultures that contained many other cell types. In a recent publication, Gouon-Evans et al. used a combination of activin A, BMP-4 and bfgf to differentiate mouse ES cells grown on collagen into a high proportion of cells positive for -fetoprotein and albumin [97]. The cells derived in that study also have other characteristics indicative of more mature hepatocytes. Although the field of hes cell research is much younger than the corresponding mouse field, a limited number of studies have evaluated the differentiation of hes cells to hepatic cell types. It was early shown that hes cells has the potential to spontaneously differentiate into such cells [41,98],but soon thereafter some groups reported on more directed differentiation. Rambhatla and co-workers reported in 2003 that hes cells differentiated with or without EB formation, in the presence of 5 mm sodium butyrate and 1% DMSO, will form cells with hepatocyte-like morphology [99]. No differences in maturation state could be identified if the EB or the direct differentiation protocol was used, and the resulting cell types were found to express hepatocyte-associated markers, such as albumin, -1-antitrypsin and cytokeratin (CK) 8 and 18. The hepatocyte-like cells derived in this study furthermore accumulated glycogen and showed inducible activity of one of the many cytochrome P450 (CYP) enzymes, CYP1A2, measured with the ethoxy resorufin O-de-ethylase (EROD) assay. One year later, Lavon et al. used a reporter gene construct regulated by the albumin promoter to isolate hepatocyte-like cells from human EBs [100]. The egfp-positive cells could be sorted using FACS and maintained in culture for a few weeks. The authors used conditioned media from cultures of primary human hepatocytes to induce differentiation of hes cells to a hepatic cell type producing albumin, and moreover identified acidic FGF (afgf) as a single factor with positive effect on these processes. However, in these two first studies only limited data on the characteristics of the hepatocytelike cells were disclosed. In another study, addition of FGF-4 and hepatocyte growth factor (HGF) induced formation of hepatocyte-like cells on collagen I or Matrigel coated dishes [101]. This group studied expression of hepatic transcription factors, such as Forkhead Box A2 (Foxa2, also called HNF-3 ), HNF-1 and GATA-4. The cells were able to produce urea and albumin, and were able to take up indocyanine green, the latter suggesting functional transporter systems. Notably, if the hepatocyte-like cells produced in this study were cultured for 4 days with Phenobarbital, a significant increase in pentoxyresorufin (PROD) activity was detected suggesting the expression of functional CYP2B6 enzyme. The disadvantage with this differentiation system was the low efficiency, with a yield of only about 2% of the cells positive for albumin and CK18. In an interesting study by Baharvand et al., the hepatic-potential of hes cell-derived EBs in 2D and 3D collagen culture systems were compared [102]. To induce hepatic differentiation, several factors were added to the growing cells, including afgf, HGF, dexamethasone and oncostatin M. Although both systems gave rise to cells with hepatocyte-like morphology and phenotype, the 3D culture appeared to have kinetic advantages. The yield of albumin and CK18 positive cells was approximately 50% in both the 2D and 3D culture system. It was unclear from this study, however, if functional systems important for drug metabolism and toxicity in hepatocytes, e.g. the phases 1 and 2 enzymes, were expressed in the two cell populations. In a very recent study, Soto-Gutierrez and colleagues cultured EBs on poly-amino-urethane coated polytetrafluoroethylene fabric [96]. The use of bfgf, a deletion variant of HGF, dexamethasone and 1% DMSO led to differentiation of hes cells to progenies with hepatocyte-like morphology. These cells produce albumin and urea, and were able to metabolize ammonia. Importantly, the authors also indicated that lidocaine was metabolized by the cells. This is the first data showing drug-metabolizing effects of any hes cell-derived hepatic cells. Data from our own lab show the capacity of hes cells to differentiate into both hepatoblast- and hepatocyte-like cells in a two-dimensional culture system (Fig. 4). After about 2 weeks in culture using a specific protocol, cells resembling hepatoblasts are developed. After about one additional week, these cells have been shown to mature further into cells with clear hepatocyte-like morphology. The yield of these cells is about 50%. Moreover, these cells are positive for many mature liver markers and express functional systems and liver transporters like bile salt acid pump and organic anion transporter proteins (Biochemical Pharmacology, accepted for publication). For future industrial use of stem cell-derived hepatocytes, the expression of specific biotransforming enzymes in the cells are of outmost importance. In a directed study we have therefore addressed this issue by Fig. 4. Hepatocyte-like cells derived from hes cells. Human ES are differentiated in vitro and after about 2 weeks, cells resembling hepatoblasts are developed. These cells have been shown to mature further into cells with hepatocyte-like morphology after approximately one additional week. These cells are positive for many mature liver markers and they express functional systems like phases 1 and 2 enzymes as well as liver transporters such as bile salt acid pump and organic anion transporter protein.

9 62 C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) studying expression and activity of the some important phase 2 enzymes. It was shown that the activities of these enzymes closely resemble those of human hepatocytes [103]. It should also be noted that the use of a direct differentiation protocol is a great advantage compared to the involvement of aggregation cultures, since it is likely to be much more amenable for large-scale production of cells for industrial applications. It should be made clear that there is no strong evidence in any of the described papers whether the hepatocyte-like cells differentiated from hes cells are derived from definitive endoderm or not. A huge hurdle in the field is the limited availability of reliable markers for definitive endoderm in human tissue. Another hurdle is that hes cells are very hard to stably transfect, something that makes the establishment of efficient reporter lines tricky. A recently published paper by D Amour and colleagues brings, however, new light into the definitive endoderm issue [104]. Most likely this study will attract more focused efforts to derive hepatocyte-like cells from a starting material consisting of pure definitive endoderm. In fact, in a recent study the derivation of hepatocyte-like cells from definitive endoderm, was reported [105]. In this protocol, hes cells were induced by activin A, and further treated with FGF-4 and BMP-2. The resulting cells showed expression of hepatic genes and the presence of protein-markers in addition to exhibiting functions similar to adult liver cells. However, no metabolism, biotransformation or transport of pharmaceutical compounds was reported. In our own laboratories, we have taken a slightly different approach for exploiting activin A induction of definitive endoderm and subsequent derivation of hepatocyte-like cells (G. Brolén and N. Heins, unpublished results). It will be interesting to ascertain the further differentiation of the definitive endoderm cells towards the hepatic lineage. If this will lead to creation of more functional hepatic cells is however still an open question. Different groups, including our own lab, have used various protocols to derive hepatocyte-like cells and the level of characterization performed varies substantially. Some key issues have been studied, but conclusive data are still lacking for culture technique (direct differentiation versus aggregate culture), culture media including factor supplementation, and surface composition (e.g. feeder cells, collagen/matrigel or bioartificial fabrics). Another very challenging future aspect is maturation of cells in bioreactors giving improved possibilities to mimic the cell cell signaling and physical parameters that are affecting hepatocytes during human liver development. In conclusion, no study so far has been able to claim the presence of high quality hepatocytes differentiated from hes cells that is derived with a protocol that could be used for industrialized applications. Even more important, it remains to be further investigated that a population of such hepatic cells is functional enough to be used for broad drug discovery and toxicology applications. Critical functions in this respect are metabolic competence, biotransformation capacity and transportation of exogenous compounds. Taken together, much more data on hes cell-derived hepatocyte-like cells are needed before we can state any such cell population as fully functional. Since the derivation of functional hepatocytes from hes cells with a scalable method will meet a huge industrial need, we will most likely see many studies addressing these and related issues in the near future Cardiomyocytes Cardiogenesis The earliest events of organogenesis during embryonic development are the formation of the heart and the initiation of its functions. Although the knowledge about the molecular mechanisms that govern cardiogenesis in humans is still in its infancy, experimental animal models have been very useful for identifying possible key regulators of heart formation. The initial signals that recruit cells to a cardiogenic fate are part of the process that patterns the early embryo [106]. In particular, the endoderm appears to have a directive function for cardiogenesis in the developing fetus [107]. The induction of cardiogenesis is characterized by the expression of transcription factors such as members of the GATA family of transcription factors, Nkx2.5, Mef2C, Tbx5/20 and Hand1/2 [ ]. By initiating the complex myocardial cross regulatory network these factors are believed to be involved in morphogenic events leading to the formation of the four chambered heart [113,114]. In addition, there are several signaling pathways and growth factors which have been implicated in early embryonic heart formation. Among the most studied are Wnts/Nodal, BMPs and FGFs [ ] ES cell differentiation to cardiac myocytes In ES cell differentiation cultures, the development of the cardiac lineage is easily detected among other differentiated cell types using light microscopy by the appearance of spontaneously contracting areas of cells. It has now been more than 20 years since the first study reported on the capacity of mouse ES cells to form EBs in suspension with subsequent development of myocardium [119]. Further studies using mouse ES cells in the early 1990s indicated the usefulness of these cells for recapitulating cardiogenesis and the development of the cardiac contractile apparatus in vitro [ ]. In addition to mouse ES cells, studies on P19 mouse embryonal carcinoma cells have provided opportunities for in vitro modeling of cardiomyocyte differentiation [124,125]. Although many factors and pathways controlling cardiac differentiation have been identified in the mouse and other model organisms (e.g. chick, xenopus and drosophila), few have been successfully verified in the human cell system. The apparent lack of overlap may reflect general differences between the experimental systems as well as fundamental intrinsic differences between the species. Notably, the kinetics of the differentiation of ES cells to cardiomyocytes is different between the murine and human models. In the mouse system, spontaneously beating clusters of cells appear 1 day after plating of EBs and within 10 days the vast majority of the EBs contain beating foci [123]. In the human cells, spontaneous beating generally commences 4 days after EB plating and new areas can

10 C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) appear up to about 30 days post-plating [ ]. This is, however, not unexpected since the initiation of differentiation by myocardial and endocardial precursors which leads up to the formation of the cardiac valves normally takes 12 days in a mouse embryo compared to 35 days in the human analogue [129]. In any case, these observations underscore the importance of performing studies using human cells in order to further our understanding of the molecular events controlling cardiac development in humans. Traditionally, the most common way to obtain spontaneously contracting cardiomyocytes from hes cells has been to differentiate the cells via EBs (Fig. 5) [41,127]. However, the efficiency of cardiogenesis in hes cells has been reported to vary and 5 70% of the EBs give rise to contracting cardiomyocytes. This broad frequency distribution probably reflects variations of the culture conditions used and inherent differences between hes cell lines. Investigators have worked intensively over the last years to develop more efficient systems for converting ES cells into cardiomyocytes relying on lessons from studies of the developing embryo. In mouse and avian embryos it has been reported that primitive streak and visceral endoderm are important for the processes that direct cardiac progenitors towards terminal differentiation [ ]. In an attempt to mimic this situation in vitro, a co-culture system was developed in which hes cells were cultured together with mitotically inactivated END-2 cells (a visceral endoderm cell line) in order to support cardiac differentiation [133,134]. However, the END-2 factor(s) promoting cardiac differentiation of hes cells are still unknown. Besides the BMP-, Wnt- and FGF signaling pathways, additional components, such as ascorbic acid, nitric oxide, Cripto, SPARC, cardiogenol and S100A4, have been shown to promote or improve cardiomyocyte differentiation in ES cells cultures [ ]. Whether these factors have direct effects on cardiogenesis or if they stimulate certain other cell populations which in turn activate or inhibit cardiac development remains to be determined. DNA methylation also appears to be of importance during cardiomyocyte differentiation of hes cells and the demethylating agent 5- aza-deoxycytidin has been reported to stimulate formation of beating cells in differentiating human EBs [141,142]. However, this effect was critically depending on the concentration and timing of administration. In line with this observation, Noggin (a BMP antagonist) was reported to induce cardiomyocyte differentiation of mouse ES cells in a restricted and time dependent fashion [143]. Taken together, it is likely that parameters such as dose, timing, isoform and combination of factors will require certain attention. Fig. 5. Morphological illustration of hes cells differentiating to cardiomyocytes. Undifferentiated hes cells were maintained in serum containing medium for 6 days in suspension cultures to form EBs. The EBs were subsequently plated in gelatin-coated cell culture dishes leading to attachment and further differentiation of the cells. Panel A shows an EB 1 day after plating in a culture dish. Panel B shows spontaneously beating areas present in the outgrowth of an EB 6 days after plating (dashed circles). Panel C shows a mechanically isolated beating area sub-cultured for 3 days after isolation in a new culture dish. Panel D shows isolated and enzymatically dissociated single cardiomyocytes derived from hes cells. Spontaneously contracting cells are indicated (arrows).

11 64 C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) To investigate the molecular program involved in early cardiogenesis, the global gene expression profile of hes cells differentiating into cardiomyocytes was recently analyzed [144]. This study employed the co-culture system of END-2 cells cultured together with hes cells in serum-free conditions [145] and the investigators harvested differentiating cells at discrete time points. Subsequent cluster analysis identified genes and signaling pathways important for cardiogenesis (e.g. Notch- and Wnt signaling). Furthermore, several transcription factors, such as Mef2C, Tbx2 and Tbx5, known to be involved in cardiac development, were upregulated during differentiation and this paralleled the expression of other known cardiac genes. In addition, this study began to identify novel genes that could potentially be important regulators during cardiac lineage specification. Two such examples are SRD5A2L2 and SYNPO2L, and whole mount in situ hybridization on mouse embryos showed cardiac restricted expression of the mouse orthologs of these genes [144]. Although the expression patterns of these genes indicate an association with heart formation, additional studies are necessary to confirm their functional contribution to this process. In addition to key signaling and transcriptional regulation of the cardiomyogenic program, recent observations have suggested a role for muscle specific micrornas that are involved in the differentiation of cardiac precursors in the embryo [146,147]. Potentially, modulation of the levels of specific micrornas could be a novel approach for initiating or sustaining cardiogenesis. An alternative approach could be to overexpress transcriptional regulators that affect the differentiation process and direct the cells into the cardiac lineage. In this regard, overexpression of GATA-4 in P19 embryonal carcinoma cells led to accelerated cardiogenesis and an increased number of spontaneously contracting cells following initiation of differentiation [148]. It remains to be determined whether these approaches will prove useful also for hes cells. One of the major obstacles for the utilization of hes cellderived cardiomyocytes (hes-cm) is the insufficient number of cells achieved by the currently described differentiation protocols. Further studies will hopefully determine additional mechanisms and factors involved in cardiogenesis and help to define the regulatory network involved in the formation of the heart. The delineation of this complex process will allow rational design of novel approaches in which the differentiation of hes cells to cardiomyocytes can be more efficiently directed Characteristics of hes-cm During recent years, cardiomyocytes derived from hes cells have been characterized on the molecular, structural and functional level. Although substantial heterogeneity has been described, in general 30 60% of the cells in isolated beating areas display markers and features of cardiac myocytes [127,149,150]. The histological appearance of hes-cm is similar to their mouse counterparts and cells with different morphologies are observed including round, small, elongated, branched and triangular cells with one or two oval nuclei and prominent nucleoli [151,152]. In contrast to the mouse ES-CM, the human analogues only display multinucleation at a very limited frequency (<1%) [150,153]. This should be compared with the fraction of bi- or multinucleated adult human cardiomyocytes that is about 20% [154]. Mature cardiomyocytes also have a more defined rod shape compared to what is usually observed in cultures of hes-cm where mixtures of individual cells display different morphologies [127,141,155]. On an ultrastructural level, transmission electron microscopy indicated that the hes-cm in early stage contracting areas in general had more disorganized myofibrillar stacks compared to cells isolated from later stage differentiated cells. Notably, the hes-cm contained Z-band, gap-junctions and granules of ANP [127,142,155]. Although several studies have demonstrated the expression of structural elements in hes-cm, the myofibrillar and sarcomeric organization indicate an immature phenotype. Complicating the interpretation and comparison of the data across studies is the fact that most investigators have analyzed the hes-cm at different stages of differentiation. In an attempt to address this issue a recent report investigated the in vitro maturation of hes-cm using electron microscopy [150]. By defining various stages of differentiated hes-cm from early (10 20 days), via intermediate (20 50 days) up to late stage (>50 days) cells the authors could describe a progressive organization of the sarcomeric pattern. Z-bands were observed starting from the intermediate phase and at the later stage also discrete A and I bands were identified in the sarcomeres. Although the level of maturity of adult cardiomyocytes is not achieved, these results suggest the possibility to modulate maturation in vitro using various stimuli (e.g. mechanical or chemical). Using standard laboratory techniques, several studies have documented that hes-cm express a number of important cardiac markers (Fig. 6). Expression of transcription factors involved in cardiogenesis such as GATA-4, Nkx2.5, Isl-1 and Mef2c is typically observed also in the hes-cm [127,141,142,145,155,156]. In addition, structural proteins including -MHC, -MHC, ctnt, ctni, MLC-2A, MLC- 2V, -actinin, desmin and tropomyosin are also expressed [126,133,141]. Notably, the staining patterns of the structural elements range from cytoplasmic clumps to parallel bundles of fibrillar structures in different cells, most likely reflecting the variation of maturation of the hes-cm. Creatine kinase-mb and myoglobin expression suggests that the cells have metabolic activity indicative for myocytes [141]. Additional cardiac markers such as ANP, connexin 43 and connexin 45 have also been detected by several independent groups [141,149,156]. In vitro functionality of hes-cm has been examined using different pharmacological and electrophysiological approaches. Cardiomyocytes derived from hes cells typically have a beating rate of about beats/min and

12 C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) Fig. 6. Immunohistochemical analysis of hes cell-derived cardiomyocytes. Human ES cells were differentiated in vitro into spontaneously contracting cardiomyocytes. Contracting areas were mechanically isolated, enzymatically dissociated into single cell suspensions and re-seeded in new culture dishes. Immuno-labeling of the cells indicates expression of (A) Nkx2.5, (B) GATA-4, (C) ctni and (D) ANP. The nuclei were counter stained using DAPI (blue). they can be maintained in vitro for several weeks, sometimes even months, without loosing functionality. In order to obtain positive or negative chronotropic responses promptly following drug application the cells need to express specific surface membrane receptors coupled to a signaling pathway that activate ion channels, membrane transporters and myofilament proteins. Cardiac pacing is accelerated through adrenergic stimulation [157] and several studies have demonstrated that functional - and -adrenergic receptors as well as muscarinic receptors are expressed in hes-cm [127,141,155,158]. Using intracellular electrophysiological measurements, nodal-like, embryonic atrial- and ventricular-like action potentials have been identified in hes-cm [126,133,141]. In addition to the initiation of action potentials in hes-cm, voltage-gated Na + channels are essential for efficient conduction of action potentials through the functional syncytium. Consistent with the functional significance of Na + channels in hes-cm, Na V 1.5 gene expression was detected by RT-PCR [159] and activities of voltage-gated Na + and K + channels were observed using whole-cell patch clamp analysis [142]. As a complement to patch clamp analysis, modulation of the electrophysiological properties by -adrenergic and muscarinic receptors in hes-cm can be studied using a micro electrode array (MEA)-system, which allows non-invasive measurements of frequency modulation as well as ion channel regulation [127,128,149]. Interestingly, the electrogram recorded from hes-cm differentiated for days was observed to be similar to the ones recorded from cultured neonatal rodent ventricular myocytes [160]. Electrophysiological maturation was observed and the QRS amplitude, QRS dv/dt and conduction velocity increased between day 1 and 3 after plating of the beating cells on the MEA. Negative force frequency relations and investigations of the Ca 2+ handling machinery using specific inhibitors suggested that the hes-cm contractions depend only on extracellular Ca 2+ and not on release of Ca 2+ from the sarcoplasmic reticulum [160]. The dysfunctional sarcoplasmic reticulum may be explained by the lack of expression of phospholamban and calsequestrin in hes-cm [161]. Interestingly, in vitro differentiated hes-cm engrafted and formed an electrical syncytium with quiescent neonatal rat ventricular myocytes and the excitation-contracting coupling was blocked chemically by 2,3-butanedione monoxime and heptanol or mechanically by physically separating the hes-cm from the rat myocytes [158]. Taken together, these results suggest that in vitro developed hes-cm are functional in that they respond appropriately to a number of pharmacological agents as well as display basic electrophysiological features. However, the hes-cm have an embryonic phenotype and they appear to have an immature sarcoplasmic reticulum function. Additional research is needed to find novel approaches to mature hes-cm in vitro.