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In Vitro Differentiation Group, IPK Gatersleben, Gatersleben, Germany; and Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Maryland
ABSTRACT I. INTRODUCTION II. PROPERTIES OF UNDIFFERENTIATED EMBRYONIC STEM CELLS A. Mouse ES Cell Lines B. Human ES Cell Lines C. ES Cells of Other Species III. GENETIC MANIPULATION OF EMBRYONIC STEM CELLS A. Random Transgenesis B. Gene Targeting IV. IN VITRO DIFFERENTIATION POTENTIAL OF EMBRYONIC STEM CELLS A. Ectodermal Differentiation B. Mesodermal Differentiation C. Endodermal Differentiation D. Germ Cell Differentiation V. EMBRYONIC STEM CELLS AS CELLULAR MODELS IN DEVELOPMENTAL BIOLOGY AND PATHOLOGY A. Gene Trapping B. In Vitro Models to Study Embryonic Lethality C. Developmental and Disease Models D. Recent Advances 1. Extrachromosomal expression 2. Recombineering 3. RNA interference VI. EXPRESSION PROFILING OF EMBRYONIC STEM CELLS A. Microarrays B. Serial Analysis of Gene Expression C. Proteomic Analyses VII. USE OF EMBRYONIC STEM CELLS IN PHARMACOLOGY AND EMBRYOTOXICOLOGY VIII. REQUIREMENTS OF STEM CELL-BASED THERAPIES A. Genetic and Epigenetic Concerns B. Tumorigenesis C. Purification and Lineage Selection D. Tissue-Specific Integration and Function E. Immunogenicity and Graft Rejection IX. EMBRYONIC STEM CELL-BASED THERAPIES A. Animal Models for Cell Therapy 1. ES cells for cardiac repair 2. ES cells used for the in vitro formation of vascular structures 3. ES cells for neurorepair 4. ES cells for the treatment of diabetes B. Therapeutic Cloning X. PROSPECTS FOR STEM CELL THERAPIES XI. OUTLOOK ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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Many of these advances are based on developmental studies of mouse embryogenesis. The first entity of life, the fertilized egg, has the ability to generate an entire organism. This capacity, defined as totipotency, is retained by early progeny of the zygote up to the eight-cell stage of the morula. Subsequently, cell differentiation results in the formation of a blastocyst composed of outer trophoblast cells and undifferentiated inner cells, commonly referred to as the "inner cell mass" (ICM). Cells of the ICM are no longer totipotent but retain the ability to develop into all cell types of the embryo proper (pluripotency; Fig. 1). The embryonic origin of mouse and human ES cells is the major reason that research in this field is a topic of great scientific interest and vigorous public debate, influenced by both ethical and legal positions.
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II. PROPERTIES OF UNDIFFERENTIATED EMBRYONIC STEM CELLS |
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Mouse ES (mES) cell lines were first established in the early 1980s (17, 98, 108, 221, 396). Initially, this required the isolation and cultivation of preimplantation embryos (blastocysts) on mouse embryonic fibroblasts (MEFs), followed by the expansion of primary ES cell outgrowths through careful enzymatic dissociation (trypsin/EDTA) and subculture regimes (see Ref. 301). The efficiency of ES cell derivation proved strain dependent, and inbred mice, like the 129 mouse strain, demonstrated the highest rates of success for the generation of ES cells (321). Once established, murine ES cell lines displayed an almost unlimited proliferation capacity in vitro (review in Ref. 333) and retained the ability to contribute to all cell lineages. In vitro, mES cells maintained a relatively normal and stable karyotype, even with continued passaging. ES cells were also characterized by a relatively short generation time of 
12–15 h with a short G1 cell cycle phase (310).
Because the generation of ES cell lines initially required a monolayer of inactivated MEFs, it was reasoned that fibroblasts provided some critical factor(s) either to promote self-renewal or to suppress differentiation. Two groups independently identified leukemia inhibitory factor [LIF (391); identical to the "differentiation inhibitory activity" DIA (334)] as the trophic factor responsible for this activity. LIF is a soluble glycoprotein of the interleukin (IL)-6 family of cytokines, which acts via a membrane-bound gp130 signaling complex to regulate a variety of cell functions through signal transduction and activation of transcription (STAT) signaling (review in Ref. 59). These cytokines, including IL-6, IL-11, oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1), all show similar properties with respect to the maintenance of pluripotency of mES cells (57, 250). The absence of IL-6 family members, the removal of MEFs, or the inactivation of STAT3, a downstream signaling molecule of the gp130 signaling complex, promote ES cells to spontaneously differentiate in vitro (39).
Studies on hematopoietic stem cell expansion had suggested that ligand-receptor complex thresholds of soluble cytokines could be maintained by high concentrations of soluble cytokines or by cytokine presentation on the cell surface. According to this model, when a relevant ligand-receptor interaction falls below a certain threshold, the probability of differentiation is increased; otherwise, self-renewal is favored. Examination of ES cells over a range of LIF concentrations demonstrated that LIF supplementation had little effect on growth rates, but it significantly altered the probability of cells undergoing self-renewal versus differentiation (414). To further address this question, a designer cytokine (a fusion protein of sIL6/sIL-6R linked to a flexible peptide chain) called Hyper-IL-6 (HIL-6) (118) together with LIF were employed to experimentally and computationally test their capacity to sustain ES cell self-renewal. Quantitative measurements of ES cell phenotypic markers, functional assays (EB formation), and transcription factor (Oct-3/4) expression over a range of LIF and HIL-6 concentrations demonstrated a superior ability of LIF to maintain ES cell pluripotentiality at higher concentrations (
500 pM). These results supported a ligand/receptor signaling threshold model of ES cell fate modulation that requires appropriate types and levels of cytokine stimulation to maintain self-renewal (375).
Identification of cell surface and molecular markers has proven critical to define the molecular basis of stem cell identity or "stemness." It is now well established that undifferentiated mES cells express specific cell surface antigens (SSEA-1; Ref. 336) and membrane-bound receptors (gp130; Refs. 57, 250) and possess enzyme activities for alkaline phosphatase (ALP; Ref. 396) and telomerase (review in Refs. 11, 277; see Table 1). ES cells also contain the epiblast/germ cell-restricted transcription factor Oct-3/4 (268, 318). In vivo, zygotic expression of this POU domain containing transcription factor is essential for the initial development of pluripotentiality in the ICM (247). In ES cells, continuous Oct-3/4 function at appropriate levels is necessary to maintain pluripotency. A less than twofold increase in expression causes differentiation into primitive endoderm and mesoderm, whereas loss of Oct-3/4 induces the formation of trophectoderm concomitant with a loss of pluripotency (251; see Fig. 4).
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Both Nanog and Oct-3/4 are essential to maintain ES cell identity, but STAT3, following LIF activation, plays an accessory role. LIF, when applied to serum-free ES cell cultures, is insufficient to maintain pluripotency or block (neural) differentiation. In combination with bone morphogenetic protein (BMP), LIF sustains self-renewal, multilineage differentiation, chimera colonization, and germ-line transmission properties. The critical contribution of BMP is to induce expression of Id ("inhibitor of differentiation") genes via the Smad pathway. Forced expression of Id genes liberates ES cells from BMP or serum dependence and allows self-renewal in LIF alone. Blockade of lineage-specific transcription factors by Id proteins enables the self-renewal response to LIF/STAT3 signaling (410). MEK/ERK signaling is also involved in ES cell self-renewal and differentiation. Inhibition of MEK/ERK by the MEK inhibitor PD098059 inhibits differentiation and maintains ES cell self-renewal in culture, and the expression of ERK and SHP-2 is thought to counteract the proliferative effects of STAT3 and promote differentiation (review in Refs. 58, 59). It however remains currently unclear how this pathway interacts with Nanog, Oct-3/4, and LIF signaling to regulate pluripotentiality (see Fig. 4).
Finally, a recent study has implicated Wnt-signaling pathways in the maintenance of ES cell pluripotency. Wnt pathway activation by a specific pharmacological inhibitor (BIO; 6-bromoindirubin-3'-oxime) of glycogen synthase kinase-3 (GSK-3) maintains the undifferentiated phenotype in both mouse and human ES cells and sustains expression of the pluripotent stage-specific transcription factors Oct-3/4 and Nanog (314). The reversibility of the BIO-mediated Wnt-activation in hES cells also suggests a practical application of GSK-3-specific inhibitors to regulate early steps of differentiation, which may prove valuable for the derivation of cells suitable for regenerative medicine.
The ES cell property of self-renewal therefore depends on a stoichiometric balance among various signaling molecules, and an imbalance in any one can cause ES cell identity to be lost. Other molecular markers potentially defining pluripotentiality include Rex-1 (163, 304), Sox2 (16), Genesis (353), GBX2 (75), UTF1 (254), Pem (112), and L17 (303). All of these have been shown to be expressed in the ICM of blastocysts and are downregulated upon differentiation; however, they are not exclusively expressed by pluripotent embryonic stem cells and can be found in other cell types in the soma. Their potential role in maintaining pluripotentiality or self-renewal remains to be determined.
The techniques used to isolate and culture murine ES cells proved critical to the generation of human (h) ES cell lines from preimplantation embryos produced by in vitro fertilization (265, 293, 362) and after in vitro culture of blastocysts (349) (see Fig. 3). The resulting hES cells shared some fundamental characteristics of murine lines, such as Oct-3/4 expression, telomerase activity, and the formation of teratomas containing derivatives of all three primary germ layers in immunodeficient mice (295, 362). Similar to mES cells, hES cells maintained proliferative potential for prolonged periods of culture and retained a normal karyotype even in clonal derivatives (4). In contrast to mES cells, hES cells formed mainly cystic EBs (168) and displayed proteoglycans (TRA-1–60, TRA-1–81, GCTM-2) and different subtypes of stage-specific antigens (SSEA-3, SSEA-4), which were absent from mouse ES cell lines (Table 1).
Several potentially important differences exist between mouse and human ES cells. hES cells show a longer average population doubling time than mES cells [30–35 h vs. 12–15 h (4)]. With murine cells, it is possible to substitute the feeder layer of embryonic fibroblasts with recombinant LIF, which signals through the gp130 receptor subunit to activate STAT3 (see above and Fig. 4). In contrast, LIF is insufficient to inhibit the differentiation of hES cells (293, 362), which continue to be cultured routinely on feeder layers of MEFs or feeder cells from human tissues. The identity of the essential self-renewal signal(s) provided to ES cells by MEF feeder cells remains ill defined. Despite the recent finding of a functional activation of the LIF/STAT3 signaling pathways in hES cells, LIF is unable to maintain the pluripotent state of hES cells (91). The cultivation of hES cells on extracellular matrix proteins, such as Matrigel (a complex mixture of ECM proteins isolated from Engelbreth-Holm-Swarm tumor) and laminin with MEF-conditioned media (401), causes hES cells to express high levels of 
6- and 
1-integrins, which are involved in cell interactions with laminin (401). These results show that the application of extracellular matrix-associated factors can be employed to improve the culture and maintenance of pluripotent hES cells.
At the end of 2001, 70 hES lines had been established using feeder layers of mouse embryonic fibroblasts. This panel of cells, however, suffers from significant limitations, including possible murine retrovirus infections (from the feeder cells) that have rendered them inappropriate for therapeutic applications. As of December 2004, only 22 of the cell lines listed in the NIH register have been successfully propagated in vitro [see update of December 10, 2004 in (http://escr.nih.gov/)], and although 17 karyologically normal (euploid) hES cell lines derived from human blastocysts were recently generated that could be subcultured by enzymatic dissociation (87), these cells were also established on MEFs. Importantly, hES cell lines have now been cultivated both on human feeder cells to avoid xenogenic contamination (5, 295) and in the absence of feeder cells under serum-free conditions (205) as had been previously done for mES cells (411). These technological advances suggest that new hES cell lines free from potential retroviral infections will be prepared and that these cells, unlike most of those currently available, might be suitable for eventual therapeutic applications.
Although the principle techniques necessary to culture (up to 80 and more passages) and manipulate hES cells have been established [cell cloning (4), cryo-preservation (294), transfection (104), and gene targeting by homologous recombination (419)], other methods (single-cell dissociation and proliferation) are still not yet optimal. Because of the variabilities among human ES cell lines (growth characteristics, differentiation potential, and culturing techniques), it will be important to define a reliable set of molecular and cellular markers that characterize the undifferentiated pluripotent (stemness) or differentiated state of hES cells. Recent attempts to define molecular markers of undifferentiated cells, however, indicate a high degree of variability among four hES cell lines maintained in a feeder-free culture system (70) and examined after long-term culture (312).
Several properties and molecular markers of hES cells are listed in Tables 1 and 2, but it is evident that the present data do not allow an unambiguous molecular definition of pluripotent stem cell properties. The application of transcriptome profiling with proteomic analyses to ES cell lines may prove useful to define which lines and growth conditions are optimal for human ES cells in vitro (see sect. VI). This information will also be necessary to set standards for hES cell research (see Ref. 52) and to answer the question, how many ES cells are necessary for research and medical applications (for further information on properties of specific hES cell lines, their cultivation, and differentiation abilities, see Ref. 79).
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Pluripotent stem cell lines have been generated from livestock (review in Ref. 277) and model organisms, such as chicken (74, 258), hamster (97), rabbit (142, 320), and rat (51, 56, 166, 372); however, only mouse and chicken ES cells have proven capable of colonizing the germ line. Of special importance for human stem cell research is the establishment of ES cell lines from nonhuman primates [rhesus monkey (263, 363), common marmoset (Callithrix jacchus, Ref. 364), and cynomolgus monkey (Macaca fascicularis, Ref. 352)]. Monkey ES cells, characterized by typical markers of human ES and EC cells (Oct-4, SSEA-4, TRA-1–60, TRA-1–81), retain a normal karyotype and have a high differentiation capacity in vitro (187, 363). These properties may qualify these cell lines as alternative and substitute model systems for hES cell lines. Moreover, after in vivo parthenogenetic development of Macaca fascicularis eggs to blastocyst-stage embryos, a pluripotent monkey stem cell line (Cyno-1) has been established that showed all the properties of hES cells, such as high telomerase and ALP activity; expression of Oct-3/4, SSEA-4, TRA 1–60, and TRA 1–81; and the ability to differentiate into various cell lineages (377). Specifically parthenogenesis is the process whereby a single egg can develop without the presence of the male counterpart.
These results suggest that stem cells derived from parthenogenetically activated eggs may also provide a potential source for autologous therapy (in the female), thus bypassing the need for creating embryos. However, aberrant expression of imprinted genes, either increased expression of maternally imprinted genes or reduced expression of paternally imprinted genes, may limit the usefulness of parthenogenetic lines and their derivatives due to their abnormal or diminished proliferative capabilities (152).
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III. GENETIC MANIPULATION OF EMBRYONIC STEM CELLS |
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DNA can be introduced into ES cells by conventional infection, transfection, or electroporation protocols (66, 67). Random insertion events have been employed to overexpress, mutate, and tag genes in phenotype-driven screens, and the discovery that DNA (cloned or genomic) introduced into ES cell lines can undergo homologous recombination at specific chromosomal loci has revolutionized our ability to study gene function. The ability to introduce virtually any mutation into the genome following gene targeting in mouse ES cells provides a powerful approach for elucidating gene function both in vitro and in the whole animal. ES cell progeny can therefore be biased into a desired cell lineage by exposure to appropriate differentiation factors and by genetic manipulations of key developmental genes. Recent advances have shown that hES cells are also amenable to genetic manipulation, thus opening the door to genetic analysis of human development and disease in vitro (104, 202, 419).
Random transgenesis results in the indiscriminate incorporation of DNA within the genome. The use of sequences that confer antibiotic resistance (e.g., neomycin, puromycin, hygromycin, and herpes simplex virus thymidine kinase) for clonal selection or of reporter genes [e.g., green fluorescent protein (GFP/EGFP), LacZ (–galactosidase)] to identify specific cell lineages has greatly facilitated this approach both in vitro and in vivo (140). Additional constructs have been designed to overexpress transcription factors (e.g., GATA4, Twist), signaling molecules (e.g., insulin-like growth factor II, Cripto), or cellular proteins in differentiated phenotypes of myogenic (95, 278, 308), erythroid (150), pancreatic (38), and cardiomyocytic (262) cell lineages. Promoters of either viral or mammalian origin have, however, often proven inconsistent in the formation of stably expressing ES cell clones.
Retroviral vectors have been used for the delivery of genetic material into cells for over 20 years. The advantage of a retroviral system is that genetic sequences can easily, efficiently, and permanently be introduced into target cells. In fact, the first successful reports of genetic manipulation of ES cells involved retroviral vectors. These early experiments demonstrated that integrated viruses (provirus) could be transmitted through the germ line (300, 348); however, sustained transgene expression from integrated proviruses proved difficult to achieve. ES cells have high de novo cytosine methylation at CpG dinucleotides, which effectively represses gene expression regulated from viral long-terminal repeats (LTRs) (28, 171, 348). In addition, provirus gene silencing is mediated by trans-acting factors that bind to the LTRs of some viral promoters (76, 260). The lack of significant provirus transcription in ES cells and ES cell progeny have effectively limited the use of simple retroviral vectors in experiments of random transgenesis (300).
The development of more complex lentiviral vectors, based on the human, feline, equine, or simian immunodeficiency viruses (246, 255, 274, 317), offer several advantages over other retroviruses (for review of vectors, see Ref. 282). Lentiviruses infect both dividing and nondividing cells, and transgene expression is not silenced in ES cells. Pfeifer et al. (271) furthermore demonstrated that lentiviral vectors could efficiently transduce human ES cells, and subsequent analyses have shown that lentivirus infections are highly effective for the delivery of functional transgenes into human ES cells (143, 214). Importantly, transgene expression is not "shut off" during differentiation in vitro (EBs) or in vivo (teratomas), and functional transgenes can be successfully passed through the germ line without loss of expression (271). These proof-of-principle experiments, with reporter constructs, demonstrate that lentiviruses are capable of foreign gene transfer to hES cells. This is particularly important, because electroporation, which has served as the main method for the introduction of foreign DNA into murine ES cells (331, 360), adversely affects the survival of hES cells (104). Lipofection-based transfection techniques, similarly, show transfer efficiency rates in hES cells that are generally <10% (104). Lentiviral delivery of foreign DNA to hES cells therefore has significant relevance for the isolation of stably transfected hES cell clones and for the future development of gene- and cell-based therapies.
Random integration of DNA plasmid constructs containing tissue-restricted promoters has been used extensively to purify or mark cells, including neurons (210), pancreatic -cells (338), cardiomyocytes (192), and endothelial cells (220, 281); however, data from these studies should be interpreted with care. In vitro expression is not always consistent with in vivo analyses. For example, vimentin, which is usually restricted to mesenchymal cells in vivo (84, 125), is expressed in most cell types in vitro (126). The myosin light chain 2v (Mlc2v) promoter has also been used to identify ventricular chamber myocytes derived from differentiating ES cells in vitro (230), but this "specific" expression is only apt for adult rodent heart. During development, this gene is expressed in the anterior (atrial and atrio-ventricular) portions of the heart tube, and at later stages, in the caval myocardium (81, 123, 124). Since ES cell-derived cardiomyocytes are not typical of adult myocardium, the Mlc2v promoter probably cannot be used to identify purely ventricular myocytes. It is therefore essential that in vitro results be analyzed in conjunction with developmental models before deciding which ES cell progeny are most useful for cellular therapeutics. Finally, integration-dependent events can adversely affect gene expression in ES cells. As with pronuclear injection, the location of integration and the number of copies of integrated DNA can affect transgene expression. In particular, transgenes randomly introduced into ES cell lines tend to be progressively silenced, resulting in mosaic expression, heterogeneous phenotypes, or complete silencing. These limitations have restricted the use of random transgenesis in functional studies of ES cells and their progeny.
Targeting approaches that selectively modify endogenous genes have generally proven more powerful than random transgenesis in generating mutations in endogenous mouse genes. In 1987, Thomas and Capecchi (360) first showed that transfected DNA could integrate into the mES cell genome via homologous recombination. In 1989, the first report of germ-line transmission of a targeted allele was published (361), demonstrating that genetically modified ES cells could contribute in the developing mouse embryo to produce viable chimeras. Today the production of germ-line chimeras is a standard procedure for many laboratories, and the topic has been extensively reviewed in the literature (47, 179).
The ability to produce mice that carry altered genomic DNA has greatly facilitated the study of many biological processes; however, not all biological processes can be studied by gene inactivation. Gene-targeting that results in developmental arrest or embryonic lethality in vivo reflects the earliest nonredundant role of a gene and precludes analysis of function at later stages. Additionally, some genes have functions during embryogenesis that may differ from those in the adult [e.g., LIF (18, 19) and vimentin (84)]. Inactivation of these genes may lead to adaptations that preclude their functional analysis at later stages. To address these problems, a number of modifications to the original gene-targeting strategies have been developed.
Embryonic lethality can be overcome by generating conditional knock-out or knock-in ES cells and mice, which can be used to activate or inactivate a gene both spatially and temporally (243). Typically, a conditionally targeted allele is made by inserting loxP or frt sites into two introns or at the opposite ends of a gene. Expression of P1 bacteriophage-derived Cre or yeast-derived Flp recombinases in mice carrying the conditional allele catalyzes recombination (insertions, deletions, inversions, duplications) between the loxP/frt sites, respectively, to inactivate (or activate) the gene (209). By expressing Cre recombinase from an endogenous or tissue-specific promoter, the conditional allele can be recombined in a restricted lineage or cell type. The timing of recombinase expression can also be controlled using inducible expression systems (313) or viral delivery systems such as adenovirus or lentivirus (270, 328), which makes it possible to inactivate a gene in a temporal-specific fashion. This technique has been widely used in the analysis of mice, and its use in adult mice overcomes a major limitation associated with standard transgenics, i.e., the developmental consequences of inactivated genes (209). The system has also been adapted for ES cell lines, both for in vitro studies and the generation of new mouse models [e.g., allele replacement by double loxP recombination (2, 395); Fig. 5]. The use of site-specific recombination events (insertions, deletions, inversions, or duplications) can also be extended to the engineering of long-range modifications in the ES cell genome (416).
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IV. IN VITRO DIFFERENTIATION POTENTIAL OF EMBRYONIC STEM CELLS |
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Differentiation is induced by culturing ES cells as aggregates (EBs; Fig. 6) in the absence of the self-renewal signals provided by feeder layers or LIF, either in hanging drops (40, 394, 395, 398), in liquid "mass culture" (98), or in methylcellulose (390). Moreover, coculture with stromal cell line activity (i.e., of PA6 cells, Ref. 186), and recently, even adherent monolayer cultures in the absence of LIF (411) have been used to differentiate mES cells in vitro. Scaleable production of ES-derived cells can furthermore be achieved through the use of stirred suspension bioreactors with encapsulation techniques (92).
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Another model to study early events of differentiation are "early primitive ectoderm-like" (EPL) cells derived from mES cells by adherent culture in medium conditioned by human hepatocellularcarcinoma HepG2 cells (MEDII-CM) (288, 289). EPL cells exhibit many properties consistent with embryonic primitive ectoderm, but are distinct from ICM and ES cells (compare Tables 1 and 2 with Fig. 1 of Ref. 302). The cells do not participate in embryogenesis following blastocyst injection. But, EPL cells allow modeling of early differentiation events without genetic modification. The aggregation of EPL cells into EBs results in a loss of visceral endoderm and neuroectoderm differentiation, whereas late primitive ectodermal, parietal endodermal, and mesodermal cells develop (302). This pattern suggests that the EPL-EB differentiation model may be suitable for studying mesoderm development in vitro and that failure to appropriately form visceral endoderm in EPL-derived EBs is responsible for the lack of ectoderm lineage formation. The defect in ectoderm differentiation, however, can be achieved by culture of EPL-EBs in the presence of MEDII-CM, which results in the formation of neuroectoderm (primitive ectoderm, neural plate, and neural tube) and an almost complete inhibition of endodermal and mesodermal differentiation (287) (see also sect. IVA).
hES cells differentiate when removed mechanically ("cut and paste") or by enzymatic dissociation from feeder layers and cultured as aggregates in suspension. Cystic EBs formed under these conditions are heterogeneous and express markers of various cell types, including those of neuronal, cardiac, and pancreatic lineages (168, 293, 323; Table 4). However, none of the factors known to influence mES cell differentiation directs hES cells exclusively into a single cell type (323). For instance, activin-A and transforming growth factor (TGF)- were found to induce mainly mesoderm; retinoic acid (RA), epidermal growth factor (EGF), BMP-4, and basic fibroblast growth factor (bFGF) elicited both ectodermal and mesodermal differentiation; whereas nerve growth factor (NGF) and hepatocyte growth factor (HGF) promoted differentiation of hES cells into all three primary germ layers. Interestingly, BMP-4 induced hES cells to develop into extraembryonic, trophoblast-like cells (403), a property clearly different from mES cells.
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Among the various lineages produced by the embryonic ectoderm during normal mouse development, the neuroectodermal lineage gives rise to the peripheral and central nervous systems (review in Ref. 212) and to the epithelial lineage, which is committed to becoming epidermal tissue (review in Ref. 130). Vascular smooth muscles are also partially derived from embryonic ectoderm.
Epithelial cell differentiation from ES cells can be identified by the presence of cytokeratin intermediate filaments and keratinocyte-specific involucrin (20, 367). After in vitro differentiation of mES cells, enrichment of keratinocytes and seeding onto various ECM proteins in the presence of BMP-4 and/or ascorbate promotes formation of an epidermal equivalent, which is composed of stratified epithelium (when cultured at the air-liquid interface on a collagen-coated acellular substratum). The resulting tissue displays morphological patterns similar to normal embryonic skin. The cells express late differentiation markers of epidermis and markers of fibroblasts, consistent with those found in native skin. The data suggest that ES cells have the capacity to reconstitute in vitro fully differentiated skin (86).
Of specific importance with regard to cell therapies of neurodegenerative disorders are neuronal and glial cells. The differentiation of mES cells into neuronal cells was published independently by three groups in 1995 (22, 122, 350). The spontaneous differentiation of ES cells into neuronal cells was rather limited (see Ref. 350) but has improved significantly by a number of strategies, involving the use of RA (review in Ref. 306), lineage selection (210, 411), and stromal cell-derived inducing activity (for review, see Refs. 141, 186). Whereas high concentrations of RA originally promoted efficient neuronal differentiation, characterized by the expression of tissue-specific genes, proteins, ion channels, and receptors in a developmentally controlled manner (122, 350), the survival and development of neurons derived in response to RA is limited. Furthermore, the teratogenicity of RA (see Ref. 306) makes it unsuitable for therapeutic applications. For these reasons, alternative protocols, involving multiple steps of differentiation and selection of neural progenitor cells, have been established. Following EB formation, serum is withdrawn to inhibit mesodermal differentiation. The proliferation of neural precursor cells is then induced by the addition of bFGF and EGF. Thereafter, neuronal cell differentiation can be supported by the addition of neuronal differentiation factors (22, 253) and maintained in vitro by neurotrophic differentiation (206) and survival-promoting factors. These include the glial cell line-derived neurotrophic factor (GDNF), neurturin (NT), TGF-3, and IL-1 (311). Significant improvements in the generation and in vitro survival of dopaminergic neurons have been achieved using these factors. Neurons can also be generated from mES cells by RA treatment combined with the genetic selection of lineage-restricted precursors (see Ref. 210), by using EPL-derived EBs in the presence of MEDII-CM (287), or by the cocultivation of ES cells with PA6 stromal cells in serum-free medium (186). In the latter case, the stromal cells produce an inducing activity, which efficiently activates neuronal differentiation, including dopaminergic cells.
Gene expression and electrophysiological studies of cell derivatives indicate the presence of all three major cell types of the brain: neurons [dopaminergic, GABAergic, serotonergic, glutamatergic and cholinergic neurons (22, 116, 122, 186, 206, 311)], astrocytes, and oligodendrocytes (8, 366; see Table 3). An elegant genetic approach to identify and validate ES cell neural regulatory genes was recently described (14). In these experiments, the earliest known specific marker of mouse neuroectoderm (early neural plate and neural tube), Sox1, was targeted with a construct containing GFP. In Sox1-GFP positive ES cell progeny, fluorescence was observed only in early neural precursors. This strategy provided a robust quantitative assay for early steps in neural differentiation. By then using an episomal expression system (see sect. VD) for uniform expression of candidate cDNAs in RA-induced ES cell derivatives, the authors identified one gene, sfrp2, that could strongly stimulate the production of neural progenitors. SFRP2 is an extracellular antagonist of the Wnt family of signaling proteins. Transfection of ES cells with Sfrp2 resulted in enhanced neural differentiation in response to RA (and neural differentiation was obtained even in the absence of RA). Overexpression of Wnt-1 in ES cells inhibited neural differentiation, thus confirming a role of Wnt signaling in ES-derived neuronal differentiation (90, see also Ref. 307). Recently, the authors went on to show that for efficient differentiation into the neural lineage, neither multicellular aggregation nor coculture is necessary. In these experiments, targeted Sox1-GFP ES cells cultured in adherent monolayers, following an efficient neural differentiation regime (N2/B27 medium) and sorting by FACS, differentiated into a highly enriched Sox1-GFP fraction of neural progenitor cells. These selected cells were further differentiated into specific neuronal, glial, and oligodendrocytic cell types (15).
The ability of human ES cells to generate derivatives of the neural epithelium was demonstrated soon after their isolation (362); however, the selective derivation of a given neuron subtype (e.g., dopamine neuron fate) had, until recently, been unsuccessful. Neural progenitor cells derived from hES cells (292) may be specifically enriched (69) and directed to differentiate into mature neurons (e.g., dopaminergic, GABAergic, serotoninergic), astrocytes, and oligodendrocytes (69; see Table 4). Growth factors, mitogens (such as RA, NGF, bFGF, and EGF) (322), ECM proteins (Matrigel, laminin; Ref. 401), and stromal cell lines (MS5, S2) as well as Wnt1-expressing stromal cells (MS5-Wnt1; Ref. 266) all serve as potent enhancers of neuronal differentiation from hES cells. Coculture of hES cells on MS5 stroma and exposure to differentiation factors, such as FGF8, SHH, and BDNF, leads to efficient differentiation of neuroepithelial structures termed "neural rosettes." Replating of these rosettes followed by terminal differentiation produces midbrain dopaminergic neurons that express the neuronal transcription factors Pax2, Pax5, and engrailed-1; release dopamine; and show characteristic properties of dopaminergic neurons by electrophysiological and electron microscopical methods. High-yield dopaminergic neuron derivation was confirmed for both human and monkey ES cell lines (266). The availability of unlimited numbers of midbrain dopaminergic neurons represents a first step towards exploring the potential of hES cells in animal models of Parkinson's disease.
Mesoderm is the germ layer that develops into muscle, bone, cartilage, blood, and connective tissue. Blood and endothelial cells are among the first differentiated mesodermal cell types to form in the developing vertebrate embryo at around day E6.5, leading to the formation of yolk sac, an extraembryonic membrane composed of adjacent mesodermal and primitive (visceral) endodermal cell layers, which give rise to blood and endothelial cells (review in Ref. 26). Hematopoietic cells and blood vessels are believed to arise from a common progenitor cell, the "hemangioblast." As with ectodermal lineages, cultured ES cells have been successfully used to recapitulate these mesodermal developmental processes in vitro. Differentiation of ES cells in complex cystic EBs permits the generation of blood islands containing erythrocytes and macrophages (98), whereas differentiation in semisolid medium is efficient for the formation of neutrophils, mast cells, macrophages, and erythroid lineages (390). Application of FCS and cytokines such as IL-3, IL-1, and granulocyte-macrophage colony stimulating factor (GM-CSF) to ES cells generates early hematopoietic precursor cells expressing both, embryonic z globin (H1) and adult major globin RNAs. Use of OP9 cells, which secrete an inducing activity, also leads to the development of all hematopoietic cell types of the erythroid, myeloid, and lymphoid lineages (244) and of natural killer (Nk) cells (review in Ref. 159). Experiments to identify potential inducers of the hematopoietic lineage furthermore indicate that Wnt3 is an important signaling molecule that plays a significant role to enhance hematopoietic commitment during in vitro differentiation of ES cells (199).
The use of endothelial cell restricted promoters illustrates how in vitro analyses of EBs can be used to define complex mesodermal-derived cells. Quinn et al. (281) used the flt-1 promoter to regulate EGFP in PECAM-1 positive ES-derived endothelial cells. The expression of this transgene, at least temporally, coincided with the expression of endogenous flt-1. Further analyses of EGFP expression relative to Sca-1 positive cells suggested that the flt-1 promoter is active in ES-derived endothelial cells, but that it is downregulated during hemangioblast differentiation to the hematopoietic lineage (281). Similarly, Marchetti et al. (220) employed the vascular endothelium-specific promoter tie-1 to drive both EGFP and pacR expression to isolate endothelial cells from genetically modified ES cells. Puromycin (pacR)-resistant cells were positive for the endothelial cell surface markers, but release from puromycin selection resulted in the appearance of -smooth muscle actin positive cells, showing that endothelial cells or their progenitors could also differentiate towards smooth muscle. Finally, the expression of vascular endothelial growth factor receptor 2 (VEGF-2, known in the mouse as fetal liver kinase 1, Flk1) in early mesodermal progenitor cells also enabled the isolation of a Flk1+ cell population that includes endothelial and hematopoietic precursors (127, 249).
A similar strategy was used to study the specification of ES cells into the "hemangioblast." ES cell lines were created that express GFP targeted to the mesodermal gene brachyury (114), a transiently expressed mesoderm-specific transcription factor (176). Analysis of brachyury-GFP targeted cells permitted discrimination between mesoderm and neuroectoderm progenitors. Coexpression analysis of GFP with FLK1, furthermore, revealed three distinct mesodermal cell populations: premesoderm (GFP-/Flk1-), prehemangioblast mesoderm (GFP+/Flk1-), and the "hemangioblast" (GFP+/Flk1+) population, the precursor cells of primitive and definitive hematopoiesis and endothelium (114).
The cellular phenotypes of ES-derived hematopoietic cells have been characterized by specific gene expression patterns and by cell surface antigens (380, 390); however, the most important definition for these cells is functional. ES cell derivatives must demonstrate long-term multilineage hematopoietic repopulating properties to be considered true hematopoietic stem cells. Early reports suggested that the repopulating ability of ES-derived hematopoietic progenitors may be restricted to the lymphoid system (236), but subsequent studies showed a long-term multilineage hematopoietic repopulating potential of ES-derived cells (160, 259).
Another mesodermal cell type that has been extensively analyzed is ES cell-derived cardiomyocytes. These cells readily differentiate from aggregates composed of initially 400–800 starting cells that form in the presence of high FCS (20%) and display properties similar to those observed in cardiomyocytes in vivo or in primary cultures. These cells 1) express cardiac gene products in a developmentally controlled manner (40, 113, 230), 2) show characteristic sarcomeric structures (146, 228), and 3) demonstrate contractility triggered by cardiac-specific ion currents and the expression of membrane-bound ion channels (40, 154, 216–218, 394). The cardiomyocytes develop spontaneously (review in Ref. 43; see Ref. 395) or could be induced by differentiation factors including dimethyl sulfoxide (DMSO) and RA (394) and small molecules, such as Dynorphin B (374) and cardiogenol derivatives (399).
Electrophysiological analyses indicate that early differentiated cardiomyocytes are typical of primary myocardium (216), which subsequently differentiate to atrial-, ventricle-, Purkinje-, and pacemaker-like cardiomyocytes (review in Ref. 154). Importantly, patch-clamp and Ca2+ imaging techniques have permitted a thorough temporal-dependent analysis of electrical activity and the dynamics of ion channel expression and signaling cascades during cardiomyogenesis (1, 167, 174, 227). Microelectrode arrays (MEA) have furthermore been employed to temporally analyze excitation generation within ES-derived cardiac clusters. When EBs are plated onto MEAs, the electrical signals of the field potentials can be recorded over a period of several days from a multitude of electrodes beneath the spontaneously contracting cardiac clusters (24).
Cardiomyocytes differentiated from hES cells show similar properties to those derived from mES cells. Cardiac clusters have been identified on the basis of spontaneous contractions. The cell clusters are composed initially of small mononuclear cells with round or rod-shaped morphology that progress to form highly organized sarcomeric structures at later stages. The cardiac-specific gene expression pattern, electrophysiological properties, and chronotropic responses to adrenergic and muscarinic agonists are also typical of cardiomyocytes (188, 239, 240, 402). Cardiomyocytes differentiated from mouse and human ES cells show similar responses to -adrenergic and muscarinic modulation (290). The differentiation protocols with hES cells, however, yield an insufficient quantity of cardiac cells for experimental analyses. In this context, the recent discovery of cardiac-inducing signals from the endoderm (239) represents a step forward to the generation of cardiomyocytes from hES cells in vitro. The authors cocultured nonbeating EBs of hES cells on a monolayer of END-2 cells, an endodermal derivative generated from P19 embryonic carcinoma cells (241). This procedure resulted in the development of functional cardiomyocytes from hES cells. The continued identification of the molecular nature of the endoderm-derived factors and the application of efficient lineage selection strategies are requirements for the derivation of cardiac tissue from hES cells.
mES cells efficiently differentiate into several other mesodermal cell types, including mesenchymal cell-derived adipogenic (93), chondrogenic (194), osteoblast (61), and myogenic (309) cells (see Table 3). In all cases, the derivation of these cell types was induced by specific differentiation factors. Although all the protocols differ, they involve the successive treatment with specific growth and matrix factors, followed by a coordinated pattern of successive steps of differentiation. A sophisticated spinner culture system has also been established to generate vascular endothelial cells useful as a murine in vitro model for blood vessel development (381). Differentiation induction of mES cells by RA and dibutyryl cAMP resulted in the development of functional vascular smooth muscle cells typical of cells found in large arteries (99). These data show that complex vascular structures, as part of the cardiovascular system, originating in vivo from both mesoderm and neural-crest lineages, can be generated from ES cells in vitro.
Pancreas and liver cells are derivatives of the definitive endoderm. During embryogenesis, the pancreas develops from dorsal and ventral regions of the foregut, whereas the liver originates from the foregut adjacent to the ventral pancreas compartment. Pancreatic and hepatic cells are of special therapeutic interest for the treatment of hepatic failure (147) and diabetes mellitus (337), and both pancreatic endocrine and hepatic cells develop in vitro from ES cells.
ES-derived hepatic cells show hepatic-restricted transcripts and proteins (149, 177) and can successfully integrate and function in a host liver following transplantation (78, 80, 404, 405). These data suggest that mES cells differentiate into all three lineages of the liver (hepatocytes as well as bile duct epithelial and oval cells). Differentiation strategies have begun to identify specific progenitor cells in the ES cell progeny, which may be of further use to isolate hepatic precursor cells of the liver (181, 182).
Hepatocyte-like endodermal markers were also detected in hES cell derivatives (285, 323). The successful differentiation and isolation of hepatic-like cells from hES cells has been demonstrated by using hES cells stably transfected with the reporter gene EGFP fused to an albumin promoter (203).
The generation of ES-derived insulin-producing pancreatic endocrine cells may be critical to the treatment of diabetes. The first successful induction of pancreatic differentiation from ES cells was obtained by stable transfection with a vector containing a neomycin-resistance gene under the control of the insulin promoter. This enabled lineage selection and maturation of insulin-expressing cells which, upon transplantation, resulted in the normalization of glycemia in streptozotocin-induced diabetic mice (338). In contrast, the spontaneous differentiation of mES cells in vitro generated only a small fraction of cells (0.1%) with characteristics of insulin-producing -like cells (329). This percentage has been increased by the selection of nestin-positive progenitor cells, the products of which showed regulated insulin release in vitro. The insulin-positive clusters, however, failed to normalize high blood glucose levels in transplantation experiments (213). Indeed, subsequent analyses revealed that these insulin-positive cells may be partially committed to a neural fate (330) and are characterized by small, condensed nuclei and are apoptotic. Rather than producing insulin themselves, most of the cells took up this hormone from the culture medium (283).
By modifying the differentiation protocols and using genetically modified mES cells, two groups successfully generated insulin-producing cells (38, 207). Blyszczuk et al. (33) showed that constitutive expression of the pancreatic developmental control gene Pax4 and histotypic differentiation were essential for the formation of insulin-expressing cells, which were found to contain secretory granules typical of both embryonal and adult -cells. Importantly, these cells coexpressed C-peptide and normalized blood glucose levels after transplantation into diabetic mice (37, 38). Similarly, lineage selection using mES cells transfected with a plasmid containing the Nkx6.1 promoter upstream of a neomycin-resistance gene could be used to generate insulin-producing cells that normalized glycemia after transplantation into diabetic animals (207).
Also, the treatment of mES cells with a phosphoinositide 3-kinase (PI 3-K) inhibitor during terminal stages of differentiation generated ES cell progeny expressing various -cell-specific markers. Following engraftment into diabetic mice, these cells also improved the glycemic status and enhanced animal survival (162).
Initial experiments with hES cells indicate that in vitro differentiation generates 1% insulin-secreting cells that show at least some characteristics of pancreatic endocrine cells (13). Treatment of hES cells with NGF results in upregulation of the Pdx-1 gene, the product of which controls insulin transcription and regulates insulin release (323). A modification of the differentiation protocol (see Refs.
