Chemical and Functional Genomic Approaches to Stem Cell Biology and Regenerative Medicine

CHAPTER 1 EMBRYONIC STEM CELLS Crystal L. Sengstaken, Eric N. Schulze, and Qi-Long Ying Center for Stem Cell and Regenerative Medicine, Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, California Embryonic stem (ES) cells are pluripotent cell lines derived from the inner cell mass (ICM) of preimplantation embryos. ES cells are invaluable tools to create genetic modifications in mice for the study of gene function and disease. The first true ES cell lines were derived from the 129 strain of mouse in 1981 by two groups simultaneously. In 1998, Thomson and colleagues reported the isolation of the first human ES cell lines from human blastocysts left over from in vitro fertilization. Putative ES or ES-like cells from other species, such as, bird, fish, monkey, dog, cow, and rat, have also been reported. However, only ES cells from mice have proved to be able to efficiently contribute to chimeras and reenter germline. There are other pluripotent cell lines including embryonic germ (EG) cells, which are derived from gonadal ridges of the embryo, and embryonic carcinoma (EC) cells, which are isolated from spontaneously arising teratocarcinomas and are therefore karyotypically abnormal. In this chapter, we will focus on mouse and human ES cells. 1.1 THE ORIGIN OF EMBRYONIC STEM (ES) CELLS There appears to be a very limited period of embryonic development during which pluripotent ES cells can be established in culture. This period is termed the preimplantation blastocyst stage (Figure 1.1). From the one-cell to early eight-cell stage, the blastomeres of the embryo are equipotent. Each blastomere is considered to be totipotent ; that is, they have the ability to differentiate into all cell types in an organism, including the extraembryonic tissue associated with that organism, and are able to form an entire organism. However, blastomeres lack the capacity for self-renewal and therefore are not considered stem cells. As cleavage proceeds, there is a gradual restriction in the developmental potency of the cells, eventually resulting in the generation of the first two distinct lineages: the trophectoderm and the ICM, followed by the formation of a fully expanded blastocyst consisting of a hollow vesicle of trophectoderm surrounding a fluid-filled cavity and a small group of ICM cells. The preimplantation blastocyst is formed after about six cleavage divisions, which occur about 3.5 or 5.5 days after fertilization in the mouse and human, respectively. The trophectoderm cells are required for implantation and development of the placenta. The ICM is the foundation of all somatic tissues and germ cells in adults. The ICM comprises the inner pluripotent primitive ectoderm cells and the outer primitive endoderm cells. ES cells are derived from the primitive ectoderm cells when ICM is isolated and cultured in vitro under proper conditions. Little is known, however, about how pluripotent primitive ectoderm cells in the ICM are transformed into pluripotent ES cells in culture. It is thought that the pluripotent ES cells remain in a state in vitro that may occur only transiently in vivo , since the primitive ectoderm cells progress rapidly through development to form the epiblast cells. Although these epiblast cells are still pluripotent and can give rise to all three primary germ layers, ectoderm, mesoderm, and endoderm, there is still no evidence suggesting that ES cells can be derived from the pluritpotent epiblast cells of the implanted embryo. However, cells resembling ES cells in both morphology and growth characteristics have been obtained previously from preblastocyst stages. Since attached embryos from progressively earlier developmental stages require longer periods in culture before they reach a state that yield ES cells, the possibility exists that preblastocyst embryos progress to an equivalent stage of blastocyst embryos in culture before ES cells can be obtained. It remains unclear, however, whether this state represents the sole timepoint from which ES cells can be obtained or whether it demarcates the beginning of a window of time during which the derivation of ES cells is possible. 1.2 DERIVATION OF ES CELLS The research on embryonic carcinoma (EC) cells in the 1970s eventually paved the way for the establishment of the first ES cell lines. When epiblast cells from early postimplantation embryos were grafted into adult mice, they produced teratocarcinomas. Teratocarcinomas are malignant multidifferentiated tumors containing a proportion of undifferentiated cells. These undifferentiated cells could be propagated in culture and established as cell lines termed EC cells . Clonally isolated EC cells retained the capacity for differentiation into derivatives of all three germ layers. EC cells could also participate in embryonic development when introduced into the ICM of blastocysts to generate chimeric mice. Maintenance of the undifferentiated state of EC cells relied on cocultivation with feeder cells, usually mitotically inactivated mouse embryonic fibroblasts (MEFs). It was reasoned that these feeder cells were providing some critical factors to sustain the pluripotency of the EC cells. Since EC cells have undergone transformation and karyotypic changes prior to establishment as cell lines, they are almost always aneuploid and are not capable of proceeding through meiosis to produce mature gametes. Following the discovery of EC cells, the next logical step was to attempt to directly isolate pluripotent cells from embryos. In 1981, two groups succeeded in establishing pluripotent ES cell lines from mouse embryos. The protocol for the derivation of ES cells is relatively simple, and most labs are still using the original protocol developed by Evans and Martin. In brief, the protocol involves plating intact embryos at the expanded blastocyst stage onto a mitotically inactivated feeder layer with either DMEMor GMEMas the basal culture medium supplemented with 10–20% fetal calf serum, 2-mercaptoethanol, nonessential amino acids, L-glutamine, and sodium pyruvate. After several days of culture, the cells from the ICM will expand to form a cell mass. After being disaggregated and replated onto fresh feeders, various types of differentiated colonies as well as colonies of a characteristic undifferentiated morphology will appear. The undifferentiated colonies can generally be expanded further to establish cell lines, now known as ES cells . The first human ES cell line was established in essentially the same method by Thomson and colleagues in 1998. Since ES cells were first created, it has become clear that different strains of mice vary considerably in their facility for derivation. The inbred 129 and C57Bl/6 strains are the most permissive; approximately 30% of 129 or C57Bl/6 embryos can be expected to give rise to ES cell lines. Most other strains rarely produce ES cell lines at all. The reason for this intriguing variability is not yet understood. The ES cell derivation efficiency can be increased by several modifications of the protocol. For example, subjecting the embryos to delayed implantation or diapause can improve the efficiency. Removal of the extraembryonic tissues from blastocysts mechanically or by immunosurgery can also facilitate the derivation of ES cells. It has been reported that, by combining the two techniques, the ES cell yield rates can be increased up to 100% for 129 and to over 50% for CBA embryos. In addition to removing the differentiative signals from the surrounding tissues of ICM, it is possible to encourage the self-renewal aspect of stem cell proliferation by preferentially inhibiting signaling pathways that promote differentiation. For example, commercially available inhibitors of the Erk-activating MEK pathway such as PD98059 have been used to promote ES cell self-renewal and improve the efficiency of ES cell derivation. ES cell lines from 129 and C57Bl/6 strain mice can also be efficiently derived under serum and feeder-free conditions with the addition of both the leukemia inhibitory factor (LIF) and bone marrow morphogenetic protein 4 (BMP4). 1.3 KEY PROPERTIES OF ES CELLS Embryonic stem cells are a remarkable cell type, mainly because of the two key properties they possess: unlimited proliferation and unlimited differentiation. ES cells can be maintained in culture for an extendedamount of time, perhaps even indefinitely. Additionally, even after many passages in culture, ES cells continue to maintain the ability to differentiate into any type of cell in the body. For this reason, ES cells are considered as pluripotent. Although there are some similarities between the various types of adult stemcells, pluripotent ES stem cells clearly differ from other adult stem cells in several ways. Adult stem cells are generally limited to differentiation into the cell types of their tissue of origin, making them only multipotent, and can be maintained in culture for only a very limited number of passages before they differentiate. However, pluripotent ES cells, when reintroduced into early-stage embryo, have the ability to reenter developmental processes and contribute to all cell lineages, including germ cells. The capacity to generate all fetal and adult cell lineages in vitro and in vivo , combined with the facility of genetic manipulations, makes ES cells a very powerful tool for molecular dissection of tissue differentiation and cellular (patho)physiology. Human ES cells also create a platform for a renewable source of differentiated cells for applications in pharmacogenomics and cell transplantation therapies. ES cells have other functionally important or unique properties, including derivation without transformation or immortalization, stable diploid karyotype (prolonged culture will increase genetic or epigenetic abnormality), clonogenic (can be grown from single cells; it has proved difficult for human ES cells), absence of G1 cell cycle checkpoint, and absence of X inactivation (in XX lines). 1.4 MAINTENANCE OF ES CELL SELF-RENEWAL When an ES cell divides, it has to decide whether to produce identical copies of itself (self-renewal) or to differentiate into specific cell types. This ES "cell fate" decision, that is, self-renewal versus differentiation, is greatly influenced by both extrinsic and intrinsic factors (Figure 1.2). 1.4.1 Extrinsic Factors in Mouse ES Cell Fate Determination LIF When ES cells are cultured on feeders in medium supplemented with fetal calf serum, they can be sustained in an undifferentiated state indefinitely while retaining the ability to give rise to all the cell types of the embryo and adult. It has been shown that conditioned media from the feeders has the same effect on ES cell self-renewal as the feeder layers. This observation eventually leaded to the identification of leukemia inhibitory factor (LIF) as the key cytokine secreted by feeder cells in supporting mouse ES cell self-renewal. Feeders lacking functional LIF gene do not support ES propagation effectively. LIF is a soluble glycoprotein and is a member of the interleukin-6 (IL6) family. LIF was initially identified by its activity to induce differentiation of M1 leukemia cells. Other members of the IL6 family, including oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin 1 (CT1), also have an effect similar to that of LIF in suppressing differentiation and sustaining self-renewal of mouse ES cells. LIF works by binding directly to a heterodimeric receptor complex containing two transmembrane glycoproteins, gp130 and the LIF-specific receptor subunit LIFRβ, bringing the associated JAKs into close proximity and causing their cross-phosphorylation and activation. A wide range of downstream effectors can be activated via gp130. These include the signal transducer and activator of transcription (STAT) 1, 3, and 5, the mitogen-activated protein kinases (MAPK), extracelluar regulated kinases (ERK) 1 and 2, and phosphoinositol-3 kinase (PI3K). In mouse ES cells LIF and other related cytokines sustain ES cell self-renewal mainly through activation of Stat3. Inhibition of Stat3 signaling directly, through the use of interfering mutant forms of the transcription factor Stat3F, provides the best evidence for an essential role of Stat3 in ES cell self-renewal mediated by LIF or related cytokines. Stat3F is a dominant-negative mutant of Stat3. Overexpression of Stat3F in ES cells resulted in induction of differentiation even in the presence of LIF, indicating that activation of Stat3 is essential to the LIF-mediated ES cell self-renewal. Studies using a chimaeric Stat3 molecule that can be activated directly by estradiol indicate that Stat3 activation is not only necessary but might be sufficient to block differentiation. Several groups have tried to identify LIF/Stat3 target gene(s) that are responsible for ES cell self-renewal, but so far it has not been very successful. Downstream target genes of Stat3 inES cells that mediate self-renewal are still largely unknown. In mouse ES cells, LIF/gp130 signaling also activates MAPK pathway. The ERKMAPKs Erk1 and Erk2 are strongly phosphorylated on stimulation of LIF. Phosphorylated ERKs then undergo nuclear translocation and modulate the activities of transcriptional regulators such as Elk, Myc, and the serum response factor (SRF). Inhibition of MAPK/ERK signaling by small molecule inhibitors PD98059 and U0126 limits the differentiation of ES cells and promotes self-renewal. This strongly suggests that while LIF/Stat3 pathway sustains ES cell self-renewal, the LIF/gp130/ERK pathway is antagonistic to it. The overall balance of conflicting activation of Stat3 and ERKs might well determine the efficiency of mouse ES cell self-renewal. Serum/BMP Feeder layers or LIF can support mouse ES cell self-renewal. However, these observations were made in the presence of fetal calf serum. In the absence of serum, LIF is not sufficient to sustain mouse ES cell self-renewal (Figure 1.2a). Instead, the ES cells will differentiate predominantly into neural phenotypes. Hence, there is another factor or factors needed in combination with LIF to achieve self-renewal. It has been demonstrated that bone morphogenetic protein 4 (BMP4) can replace the requirement for serum both during clonal propagation of mouse ES cells and during their de novo derivation. BMPs were originally isolated from demineralized bone matrix and identified as factors responsible for inducing bone formation in muscular tissues. The critical contribution of BMP4 to self-renewal is to induce expression of the negative helix–loop–helix factors, inhibitors of differentiation (Ids). Id proteins lack a DNA binding domain and are not thought capable of inducing gene transcription. They act by binding and sequestering E proteins, thereby inhibiting the E–protein–dependent transcriptional activity of basic helix–loop–helix (bHLH) factors. Constitutive Id gene expression in ES cells replaces the need for BMP4 in the media. Serum also induces Id genes via multiple pathways, including integrin engagement by extracellular matrix molecules such as fibronectin. A BMP-like factor was purified from serum that is responsible for both inhibition of myogenesis and stimulation of osteoblast differentiation in vitro . This BMP-like factor was identified as BMP4. In addition, BMP4 in serum was found to form a large complex with other molecules, resulting in potentiation of its activity. Therefore it is not surprising that ES cells cultured in serum also show appreciable levels of Id gene expression. These findings together suggest that the biological activities of serum in suppressing differentiation and sustaining self-renewal of mouse ES cells might be mediated at least in part by BMPs. Significantly, in the absence of LIF, both serum and BMP4 drive ES cells differentiation into nonneural fates (Figure 1.2a). Thus serum or BMP4 stimulation has a dual potential in ES cells, and the outcome is dictated by the presence or absence of LIF/Stat3 pathways. The ability of BMP4 to suppress differentiation and maintain ES cell self-renewal in collaboration with LIF/Stat3 is shared by other BMPfamily, BMP2 and GDF6, but not other transforming growth factor β (TGFb) superfamily members, such as TGFβ1 and activin A. Mouse ES cells can also be maintained in serum-free medium supplemented with LIF and knockout serum replacement without addition of serum or BMPs. Under these culture conditions, it was reported that activin or Nodal, but not TGF b1 or BMP4, can significantly enhance ES cell proliferation without affecting pluripotency. Although serum replacement is thought to be better defined than serum, it is a proprietary product that cannot be regarded as fully defined. In fact, it has been shown that BMP-like activity is present in serum replacement. Indeed, both BMP2 and BMP4 proteins were detected in serum replacement. (Continues...) Excerpted from Chemical and Functional Genomic Approaches to Stem Cell Biology and Regenerative Medicine by Sheng Ding . Copyright © 2008 John Wiley & Sons, Ltd. Excerpted by permission of John Wiley & Sons. All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher. Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site. Scientists believe that stem cells have the potential to repair specific tissues and to grow organs, revolutionizing the treatment of cardiovascular disease, neurodegenerative disease, musculoskeletal disease, diabetes, and cancer. A core reference for researchers, Chemical and Functional Genomic Approaches to Stem Cell Biology and Regenerative Medicine consolidates the latest information on functional genomics and chemical biology approaches that are used to study and control the fate of stem cells. It discusses new technologies and their recent applications in various areas of stem cell biology, covering: The use of both embryonic and adult stem cells, A vast array of technologies, including: genome-wide expression analysis, functional genomic profiling with arrayed cDNA and RNAi expression libraries, informatics, chemical genomics, mass spectrometry, and proteomics, The applications of advanced technologies in various areas of stem cell biology, encompassing: self-renewal, differentiation, and reprogramming of different types of embryonic and adult stem cells, as well as regeneration in model organisms, Technological tools for studying the biology of stem cells, With chapters contributed by leading authorities, this book is a hands-on reference for chemists, biologists, biochemists, bioinformaticians, clinicians, and managers involved in stem cell research. It is also an excellent text for interdisciplinary courses such as functional genomics, stem cell biology, and chemical biology Embryonic stem cells / Crystal L. Sengstaken, Eric N. Schulze, and Qi-Long Ying Adult stem cells / Lief Fenno and Chad A. Cowan Genome-wide expression analysis technologies / John R. Walker Genomic cDNA and RNAI functional profiling and its potential application to the study of mammalian stem cells / Jia Zhang ... [et al.] Chemical technologies: probing biology with small molecules / Nicolas Winssinger, Zbigniew Pianowski, Sofia Barluenga Protein characterization by biological mass spectrometry / Venkateshwar Reddy and Eric C. Peters Large scale genomic analysis of stem cell populations / Jonathan D. Chesnut and Mahendra S. Rao Exploring stem cell biology with small molecules and functional genomics / Julie Clark ... [et al.] Regeneration screens in model organisms / Chetana Sachidanandan and Randall T Peterson Proteomics in stem cells / Qiang Tian and W. Andy Tao. Halftitle......Page 2 Inside Cover......Page 3 Copyright......Page 4 Contents......Page 5 Contributors......Page 7 Color Plates......Page 10 1 EMBRYONIC STEM CELLS......Page 22 2 ADULT STEM CELLS......Page 47 3 GENOMEWIDE EXPRESSION ANALYSIS TECHNOLOGIES......Page 79 4 GENOMIC cDNA AND RNAi FUNCTIONAL PROFILING AND ITS POTENTIAL APPLICATION TO THE STUDY OF MAMMALIAN STEM CELLS......Page 102 5 CHEMICAL TECHNOLOGIES: PROBING BIOLOGY WITH SMALL MOLECULES......Page 127 6 PROTEIN CHARACTERIZATION BY BIOLOGICAL MASS SPECTROMETRY......Page 163 7 LARGE-SCALE GENOMIC ANALYSIS OF STEM CELL POPULATIONS......Page 186 8 EXPLORING STEM CELL BIOLOGY WITH SMALL MOLECULES AND FUNCTIONAL GENOMICS......Page 204 9 REGENERATION SCREENS IN MODEL ORGANISMS......Page 224 10 PROTEOMICS IN STEM CELLS......Page 240 INDEX......Page 260