b899_Chapter-02.qxd
10/12/2010
4:38 PM
Page 23
2 Molecular Principles Underlying Pluripotency and Differentiation of Embryonic Stem Cells Kyle M. Loh*, ‡, Boon Seng Soh*, ‡, Wai Leong Tam* and Bing Lim*,†
Introduction “You start out as a single cell derived from the coupling of a sperm and an egg; this divides in two, then four, then eight, and so on … The mere existence of such a cell should be one of the great astonishments of the earth. People ought to be walking around all day, all through their waking hours calling to each other in endless wonderment, talking of nothing except that cell.” Lewis Thomas (1979), The Medusa and the Snail1
Within the nascent embryo, there is a privileged population of cells unrestricted in potential that can give rise to every cell type present within the fetus — these remarkable cells are the pluripotent cells of the epiblast.2,3 There are severe responsibilities placed upon these epiblast cells; they have been entrusted with the responsibility of perfectly constructing the entire fetus. Their proper operation ensures the successful emergence of another life, whereas their dysfunction fatally compromises fetal development. These pluripotent cells are unlike any other types of cell within the body. They stand in contradistinction to the differentiated cells present within the adult body, which are rigidly fixated in their choice of lineage. What confers these cells with their unmatched potency to give rise to any embryonic cell type? What enables them to escape the lineage fixation of *Genome Institute of Singapore, Stem Cell & Developmental Biology, Singapore 138672. † Harvard Medical School, Department of Medicine and Beth Israel Deaconess Medical Center, Division of Hematology/Oncology, Boston, Massachusetts 02115, USA. ‡ These authors contributed equally to this work.
23 STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
24
10/12/2010
4:38 PM
Page 24
K. M. Loh et al.
differentiated cells? What signals can direct these cell to differentiate into all the specific cell types that populate the entire fetus? Such seemingly abstract questions are not solely of intellectual interest. From a practical perspective, pluripotent cells — such as embryonic stem cells and induced pluripotent stem cells — have unparalleled utility for regenerative medicine and developmental biology.4–9 Answers to the above questions will potentiate the therapeutic utility of pluripotent stem cells for regenerative medicine and will provide insights into some of the most challenging questions within developmental biology. Although in their relative infancy, pluripotent stem cells have already spearheaded key advances in regenerative medicine and they have also provided key insights into early embryonic development.10–12 Indeed, pluripotent stem cell-based therapies have succeeded in ameliorating a diverse spectrum of diseases in animal models, from diabetes19 to cardiac infarction,20 and human embryonic stem cell-based therapies are presently in preclinical testing for their eventual deployment to the clinic in order to aid human patients. Another recent breakthrough has been the successful usage of pluripotent stem cells to construct previously inaccessible in vitro models of complex human diseases, which has enabled the in vitro study of disease progression and has allowed for the creation of screening platforms for novel therapeutics.13–18 It is an exciting time for pluripotent stem cell technologies, and we anticipate that in the near future, their potential will be even more fully realized, thus advancing the reach of regenerative medicine and furthering developmental biology.
Dramatis Personae of the Pluripotent Stem Cell Ensemble In order to preface later chapters that focus on the utilization of pluripotent stem cells for therapeutic or research purposes, here we will briefly discuss how pluripotent stem cell lines are established from early embryos, and more recently, how pluripotent stem cell lines may also be ethically generated bereft of embryonic tissues. We introduce three of the most wellknown types of pluripotent stem cells and how they are derived: embryonic stem cells, epiblast stem cells, and induced pluripotent stem cells. During early embryonic development, the first direct progenitors of the embryo proper can be discerned from within the epiblast of the postimplantation blastocyst’s inner cell mass.3 The epiblast is the first pluripotent tissue within the nascent embryo — it is a population of
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
25
10/12/2010
4:38 PM
Page 25
Molecular Principles Underlying Pluripotency and Differentiation Table 1.
Overview of Early Embryonic Lineages
Common name
Function
A. Pre-implantation blastocyst (~E3.5) Inner cell mass Differentiates to give rise to the epiblast and the primitive endoderm Trophectoderm Forms the future placenta, which links the embryo to the uterine wall B. Post-implantation blastocyst (~E4.5) Early epiblast Differentiates into all fetal tissues; mESCs are derived at this stage Primitive endoderm Forms the future extraembryonic yolk sac, provides patterning signals Trophectoderm Forms the future placenta, which links the embryo to the uterine wall C. Post-implantation embryo (E5.5 – E5.75)a Mature epiblast Differentiates into all fetal tissues; EpiSCs are derived at this stage D. After gastrulation (E6.7 – onwards)a Definitive endoderm Forms gastrointestinal and respiratory tracts, liver, pancreas, and others Mesoderm Forms hematopoietic and connective tissues, bone, muscle, and others Definitive ectoderm Forms the nervous system, the skin, and neural crest derivatives (A) The mouse pre-implantation blastocyst at embryonic day 3.5 (E3.5) comprises two separate tissues — the trophectoderm and the inner cell mass. (B) The blastocyst implants into the uterine wall at ~E4.0, and shortly afterwards, the inner cell mass undergoes further differentiation such that at E4.5, the blastocyst comprises the trophectoderm as well as two additional tissues: the epiblast and the primitive endoderm, which are the two derivatives of the inner cell mass. The epiblast is sometimes alternatively referred to as the “primitive ectoderm” whereas the primitive endoderm is sometimes alternatively referred to as the “hypoblast”. The epiblast comprises pluripotent cells that will differentiate into all the cells present within the fetus. The trophectoderm and the primitive endoderm will later form extraembryonic tissues that sustain the embryo and supply extrinsic patterning signals. Mouse embryonic stem (ES) cell lines can be isolated from the pre-implantation epiblast at E4.5. (C) Shortly thereafter, the embryo dramatically transforms with the involution of the epiblast, thus forming the “egg cylinder” stage embryo at E5.5 — it is at this stage that the epiblast undergoes anterior-posterior patterning, which eventually fates different compartments of the epiblast to differentiate into one of the three major fetal lineages. Mouse epiblast stem cell (EpiSC) lines can be isolated from the post-implantation epiblast between E5.5 and E5.75. (D) Gastrulation, the onset of establishment of the three major fetal lineages, occurs at E6.5, with epiblast cells becoming allocated to either the definitive endoderm, mesoderm, or definitive ectoderm depending on their position along the anterior-posterior axis established within the epiblast. The mouse epiblast is still functionally pluripotent by E6.5, as transplantation of E6.5 post-implantation epiblast cells between the epiblast’s proximal and distal compartments shows that epiblast cells will contribute to differentiated cell types that are typically produced by the compartment in which it is transplanted to. The derivatives of the three major fetal lineages are described further within the text. a For the sake of brevity, we have omitted mention of the plethora of additional tissues present within the conceptus from E5.5 onwards (such as the extraembryonic tissues).
several dozen cells that will give rise to the entire fetus and all of its three principal cellular lineages; the definitive endoderm, the mesoderm, and the definitive ectoderm (summarized in Table 1).21,22 Given the capability of the pluripotent epiblast to give rise to all fetal cell types, we
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
26
10/12/2010
4:38 PM
Page 26
K. M. Loh et al.
define pluripotency as the functional capacity to differentiate into all cell types present within the fetus. Shortly after the blastocyst implants, the epiblast elongates and begins receiving regionalized patterning signals that direct different compartments of it to differentiate into the definitive endoderm, the mesoderm, or the definitive ectoderm.23,24 This initial wave of differentiation, in which discrete compartments of the epiblast are allocated to differentiate into these three lineages, leads to the expenditure of the epiblast’s pluripotential capacity. Once the epiblast’s pluripotency is exploited in order for it to generate these three major lineages, the epiblast’s pluripotency is forever lost — indeed, upon commitment to one of the three major fetal lineages, the epiblast’s differentiated progeny are largely incapable of generating cell types from the other two fetal lineages.25 Thus, the epiblast’s pluripotency is irreversibly lost as it embarks upon differentiation Although the pluripotent character of the epiblast is quickly lost during early development, it is possible to capture cell lines that are indefinitely pluripotent from the early post-implantation epiblast prior to the expenditure of its pluripotency. Explantation of the early mouse epiblast (E4.5) into specific cell culture conditions gives rise to infinitely-proliferating pluripotent cell lines, known as mouse embryonic stem (ES) cell lines; these cells, unlike their in vivo antecedents within the epiblast, retain their pluripotency indefinitely in culture.26–28 Although there are marked gene expression differences between mouse ES cells and the post-implantation epiblast from which they are derived,29 mouse ES cells are nevertheless operationally pluripotent, as they can differentiate into every embryonic cell type present within the fetus.30,31 Amazingly, when mouse ES cells are injected into tetraploid blastocysts that lack any functional pluripotent epiblast cells (a procedure known as “tetraploid complementation”), they can rescue fetal development, taking over the place of the absent epiblast and differentiating in its stead, giving rise to live and fertile mice.30,31 Given that mouse ES cells can differentiate into all cell types within the fetus just like the epiblast, mouse ES cell lines are convenient tools that allow investigators to simulate and study embryonic development in vitro bereft of embryonic tissues and without resorting to intensive embryonic micromanipulation techniques.30 Another distirct class of pluripotent cell lines, known as mouse epiblast stem cell (EpiSC) lines, can also be derived from the post-implantation epiblast at E5.5–E5.75, one day after mouse ES cells are derived.32,33 Though EpiSCs are explanted from the epiblast just like mouse ES cells are,
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
27
10/12/2010
4:38 PM
Page 27
Molecular Principles Underlying Pluripotency and Differentiation
the epiblast has undergone significant molecular changes (such as proximaldistal patterning and anterior-posterior patterning) in the one day between which ES cells and EpiSCs are derived,23,34 which are likely to be responsible for the unmistakable functional and molecular differences between mouse ES cells and EpiSCs.32,33,35,36 Although EpiSCs are capable of multilineage differentiation, they express only a subset of pluripotency genes that are typically expressed within mouse ES cells,32,33 and paradoxically, they are imbred with epigenetic features that are associated with early differentiation.35,36 The aberrant molecular nature of EpiSCs has elicited speculation that they may be more poised to differentiate than mouse ES cells2; however, for a more extensive treatment on the nature of EpiSCs, we refer the reader to recent expert reviews.2,37 Pluripotent stem cells have also been derived from early human embryos; these cells are known as human ES cells.38,39 In vitro, human ES cell lines can differentiate into cell types from all three fetal lineages.40 However, we are currently unable to inject human ES cell lines into embryos (due to ethical and technical considerations) to test whether or not human ES cells, like their mouse ES cell counterparts, can complement human development and give rise to all embryonic cell types that are present within the human fetus.6 Thus, it is currently impossible to formally prove that human ES cells are operationally pluripotent and are capable of differentiating into every single fetal cell type.41 Nevertheless, as we discuss below, human ES cells can be directed to differentiate in vitro into many cell types of therapeutic interest, so this issue is largely inconsequential to their future clinical usage. Given that all of the above three pluripotent stem cell types are derived from early embryos, the actual techniques with which they are derived have elicited formidable ethical and moral concerns, as the derivation of pluripotent stem cells from embryos often compromises the embryo and precludes its continued development.42,43 Thus, it has been hoped that these ethical concerns could be circumvented by methods that could allow for the derivation of pluripotent stem cells from embryos without compromising their future developmental potential,38,42 or better yet, to simply generate pluripotent stem cells from alternative sources besides the embryo.44 To this end, recent decisive studies have devised several pioneering approaches to ethically derive human or mouse pluripotent stem cell lines bereft of any embryonic tissues.45–49 In the most compelling of these approaches, pluripotency is bestowed upon differentiated adult cells (such as fibroblasts or keratinocytes) through the overexpression of
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
28
10/12/2010
4:38 PM
Page 28
K. M. Loh et al.
several transcription factors; this directly converts these adult cells into pluripotent stem cells known as “induced pluripotent stem cells” (iPS cells).47 Thus, pluripotent stem cells can be created from explants of any human adult’s tissues — no embryonic tissues are required. This approach to ethically generate pluripotent stem cell lines without embryonic tissues is described in detail in subsequent sections. Taken altogether, ES cells, iPS cells, and EpiSCs constitute the three most well-known types of pluripotent stem cells derived thus far from mice and humans. However, it should be noted that there are additional subtypes of pluripotent stem cells — embryonic germ cells,50,51 embryonic carcinoma cells,52,53 and germline stem cells54 — that exist but are beyond the scope of this chapter. One of the most compelling motives for why pluripotent stem cells should be studied is to exploit them for cell replacement therapies. The etiology of many human diseases, such as type 1 diabetes, lies in the deficiency or dysfunction of a specific cell type within the patient’s body, such as the pancreatic β-cell.5 Given that the defining criterion of a pluripotent cell is that it has the capacity to differentiate into every fetal cell type, a pluripotent stem cell is capable of differentiating into any cell type that is absent or dysfunctional within a patient’s body. Thus, it has been envisaged that we can direct pluripotent stem cells to differentiate into the specific relevant cell type, and then transplant these cells back into the patient, thus restoring near-normal numbers of the absent cell type and theoretically ameliorating the disease.5,55,56 This promising approach, known as “cell replacement therapy”, is a central pillar to many regenerative medicine strategies,5,56 and it already has been put into practice to ameliorate diverse diseases in animal models, such as blindness,57 cardiac infarction,20 diabetes,19 immunodeficiency,58 Parkinson’s disease,59,60 and spinal cord injury.61 Such applications in which ES cells are exploited for therapeutic gain in human patients are discussed below in brief and are reviewed more extensively in a subsequent chapter. Secondly, pluripotent cell lines are an attractive in vitro screening platform for determining the minimal extrinsic cues and growth factors that direct the development of a particular cell type during embryonic development. Given that ES cells are the operational in vitro correlates of the early in vivo post-implantation epiblast, they should differentiate into all cell types present within the fetus through molecular processes that mimic how the epiblast gives rise to all fetal cell types during organogenesis.5 Thus, identification of defined extrinsic factors that direct ES cell differentiation
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
29
10/12/2010
4:38 PM
Page 29
Molecular Principles Underlying Pluripotency and Differentiation
into a specific cell type likely defines the minimal combination of growth factors and patterning signals responsible for directing specification of that cell type during fetal development. This strategy represents a novel approach for developmental biologists to elucidate how their favorite cell types are specified during development — for example, in vitro studies with ES cells have suggested that there is a common embryonic progenitor for primitive erythropoiesis and definitive hematopoiesis.12 Furthermore, given that human embryogenesis is difficult to study due to various ethical and technical restraints, human ES cells are timely tools to model human embryonic development in vitro, thus allowing for the in vitro study of human embryogenesis without the procurement of human embryonic tissues6 — to this end, a recent study with human ES cells has implicated an unanticipated role for PKC activation in pancreatic organogenesis within humans.10 Finally, the readiness and ease with which pluripotent cell lines can be indefinitely maintained in culture makes them particularly amenable for experimental interrogation in vitro. Although the pluripotent cells of the epiblast are incapable of continued self-renewal, as they differentiate several days after their establishment,25 their in vitro immortalized counterparts (ES cells) are capable of indefinite propagation, as discussed above. The facile and robust nature of ES cells within cell culture has made them amenable to many different experimental technologies, such as gene knockout or knockin by homologous recombination,62,63 gene knockdown by transfection of interfering RNAs,64 and gene overexpression by transfection or transduction with overexpression vectors65–68 — these “gain-of-function” (overexpression) and “loss-of-function” (knockout/knockdown) approaches have been extensively exploited by investigators to elucidate which genes and signaling pathways impart ES cells with their pluripotential character, as discussed below. The enormous replicative capacity of ES cells has also made them amenable for adaptation for intensive high-throughput experimental methodologies such as high-throughput chemical screening69 and genomewide RNAi screening.70 It should also be noted that our capability to easily culture and expand ES cells in vitro sharply contrasts with our currently limited ability to maintain hematopoietic stem cells, neural stem cells, and other types of stem cells for extended periods of time in culture.71,72 Thus, ES cells are largely unique amongst most other stem cell populations in that they are convenient to interrogate in culture — in contrast, detailed gainof-function or loss-of-function studies of hematopoietic, neural, and other stem cell populations are relatively painstaking and are difficult to undertake in vitro.73
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
30
10/12/2010
4:38 PM
Page 30
K. M. Loh et al.
Having described why pluripotent stem cells are of both clinical and academic interests and how vanous pluripotent stem cell lines are derived, we now move forward to an issue of immediate interest — how ES cells can be differentiated into specific cell types of therapeutic interest. We also discuss briefly the present challenges that face the therapeutic employment of ES cell-based therapies in the clinic. In the succeeding sections, we present a treatise on the molecular programs that confer pluripotent stem cells with their pluripotent nature. In our discourse, we challenge present notions within the field and present novel hypotheses on how molecular ensembles actually function to engender pluripotency within stem cells. Finally, we discuss the recreation of pluripotency within differentiated cells — that is, directly reprogramming differentiated cells into pluripotent stem cells. We address all of these issues from a detailed molecular perspective, with the aim of defining molecular principles that underlie the differentiation and pluripotency of pluripotent stem cells. Elucidation of such principles will enable the therapeutic exploitation of pluripotent stem cells for regenerative medicine and will provide answers to paradigmatic questions in developmental biology.
Directing Embryonic Stem Cell Differentiation to Clinically Relevant Cell Types Extensive work by developmental biologists in multiple model organisms has elucidated the signaling pathways that direct the differentiation of the pluripotent epiblast into the three major fetal lineages — the definitive endoderm, mesoderm, and definitive ectoderm (summarized in Fig. 1).23,34,74 Additional work has delineated the subsequent signals that pattern these germ layers, thus specifically demarcating particular domains of each germ layer to form the progenitors of the fetal organs,75,76 and then the final signals that instruct the multipotent progenitors of these organs to differentiate into the terminally-differentiated cell types that will comprise the final organ and impart it with its variegated physiological functions.77 Before we even consider the clinical usage of ES cells in cell replacement therapies, first we must consider the following question — how shall we even differentiate ES cells into the clinically-relevant cell types that we would like to transplant? Above, we have stated that ES cells are the in vitro operational equivalent of the pluripotent epiblast, capable of differentiating
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
Figure 1. Differentiation of pluripotent stem cells to major fetal lineages. Overview of the molecular principles underlying the differentiation of pluripotent stem cells. The first lineage bifurcation available to the pluripotent epiblast is specification to the mesendoderm or definitive ectoderm. Activation of TGFβ signaling and/or Wnt signaling specifies a mesendodermal fate, whereas inhibition of TGFβ signaling specifies definitive ectoderm differentiation. The mesendodermal is the bipotential progenitor of both the definitive endoderm and the mesoderm. High levels of TGFβ signaling specifies the definitive endoderm, whereas lower levels of TGFβ signaling, BMP signaling, or Wnt signaling specifies the mesoderm. Concurrently, the definitive ectoderm is presented with the decision to either differentiate into the surface ectoderm or the neuroectoderm. Surface ectoderm differentiation is confirmed by BMP signaling, while inhibition of BMP signaling or FGF signaling specifies the neuroectoderm.
b899_Chapter-02.qxd
31
10/12/2010 4:38 PM
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
Page 31
Molecular Principles Underlying Pluripotency and Differentiation
b899_Chapter-02.qxd
32
10/12/2010
4:38 PM
Page 32
K. M. Loh et al.
into any fetal cell type. Thus, it is logical to premise that the same developmental signals that inform epiblast differentiation into specific cell types within the embryo should also instruct ES cells to differentiate into the same cell types in vitro.5,78–80 We conclude that by recreating specific patterns of differentiation signals within the embryo, we should be able to command ES cells to differentiate into specific cell types of interest in vitro. Many cell types of therapeutic interest have been efficiently generated through human ES cell, differentiation, including pancreatic β-cells,79 hepatocytes,81 cardiomyocytes,20,82 endothelial cells,83 dopaminergic neurons,84–86 motor neurons,87 oligodendrocytes,88 oligodendrocyte progenitors,61 and retinal photoreceptor cells.89 Here, we discuss informed methods used to differentiate ES cells to these lineages of interest while relying on guidance from developmental principles. The first lineage decision available to the pluripotent epiblast during development is commitment to one of the three principal fetal lineages — definitive endoderm, mesoderm, or definitive ectoderm. The differentiated cell types produced from each primary fetal lineage are generally exclusive to that lineage, although this is not always the case.90–92 The definitive endoderm gives rise to the lining of the respiratory tract, the lining of the gastrointestinal tract, the lining of the auditory tract, and certain endocrine organs (such as pancreas, liver, and thyroid gland).23,76,93 The mesoderm is the progenitor to several diverse tissues, such as the hematopoietic tissues, connective tissues, bone, and muscle,23 whereas, the definitive ectoderm gives rise to neural tissues (the neuroectoderm), the epidermis (the surface ectoderm), and neural crest derivatives (such as melanocytes).23,90,91,94 These lineage derivatives are summarized again within Table 1. How can we differentiate ES cells into the definitive endoderm, mesoderm, or the definitive ectoderm (Fig. 1)? Close characterization has revealed that the endoderm and the mesoderm actually have a common progenitor; a tissue known as the mesendoderm that has the bipotential to differentiate into both endoderm and mesoderm.95,96 Thus, resolution of the first lineage decision encountered by the epiblast results in a bifurcation in lineages, with epiblast cells committing to either to the mesendoderm (which will eventually differentiate into either the definitive endoderm or the mesoderm) or else the definitive ectoderm. This first critical decision between the mesendoderm and the definitive ectoderm appears to be largely governed by TGFβ and Wnt signaling.93,95 Treatment of ES cells with Activin A (a potent activator of
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
33
10/12/2010
4:38 PM
Page 33
Molecular Principles Underlying Pluripotency and Differentiation
TGFβ signaling) and with Wnt3a (a potent activator of Wnt signaling) leads to their efficient and rapid differentiation into the mesendoderm within 36 hours.79,93,95 The bipotential mesendoderm is a transient population that co-expresses markers specific for both the endoderm (i.e. Sox17) and mesoderm (i.e. Brachyury) — subsequent differentiation leads to the production of either definitive endoderm progenitors or mesoderm progenitors, which are distinct from one another.93,95 Differentiation of the ES-derived mesendoderm into the definitive endoderm occurs after treatment with high con-centrations of Activin A93,97,98 (Fig. 2), whereas treatment with Wnt3a, BMP4, or lower concentrations of Activin A specifies the mesoderm.97–100 The efficiency of differentiation into the definitive endoderm can further be enhanced by co-treatment with two chemical compounds (IDE1 or IDE2),101 whereas the efficiency of mesodermal differentiation can be enhanced by treatment with a chemical inhibitor of p38 MAPK (SB203580).102 After such treatments with growth factors and/or chemical compounds, definitive endoderm cells can be efficiently purified by flow cytometry sorting for cells expressing either the surface markers E-cadherin or CXCR4 — this yields a near-homogenous population of endoderm progenitors for differentiation into specific endodermal cell types of interest.93,95 Whereas mesendodermal commitment of ES cells is directed by activation of TGFβ and Wnt signaling, ectodermal specification is instructed by the suppression of TGFβ and Wnt signals. To this end, treatment of ES cells with a TGFβ inhibitor (SB-431542) leads to their differentiation to the definitive ectoderm.103 After ES cells are differentiated to the definitive ectoderm, either a neural fate (neuroectoderm) or an epidermal fate (surface ectoderm) can be specified; treatment with BMP4 elicits epidermal differentiation,104 whereas treatment with FGF4 or a BMP antagonist (Noggin) efficiently specifies neural differentiation.105,106 Neural specification of human ES cells produces multipotent neural stem cells within characteristic “neural rosettes” that are capable of differentiating into many central nervous system and peripheral nervous system cell types, such as motor neurons, GABAergic neurons, dopaminergic neurons, astrocytes, and oligodendrocytes.84 These human ES cell-derived neural stem cells can also be expanded for several passages by treatment with sonic hedgehog and the Notch agonists Jagged-1 and Dll4, thus allowing for the creation of a large pool of these stem cells, which are the progenitors to many therapeutically-applicable neural cell types.84
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
34
10/12/2010
4:38 PM
Page 34
K. M. Loh et al.
Figure 2. Directed differentiation of human ES cells to the definitive endoderm. Differentiation of human ES cells into the definitive endoderm after treatment with high concentrations of Activin A and low concentrations of serum for 5 days. (A) Quantitative PCR showed high expression of endodermal markers (Sox17, Gsc, and Foxa2) and a mesendoderm/primitive streak marker (Mixl1) as compared with other lineage markers. Gene expression values were normalized to ES cells that were cultured in mouse embryonic fibroblast conditioned medium (MEF-CM). Bars, standard deviation of n = 3 experiments. Asterisk denotes values of p < 0.05 for Kruskal-Wallis one-way analysis of variance compared to control (ES cells cultured in MEF-CM). (B) Immunocytochemistry demonstrating expression of FOXA2 and SOX17 after definitive endoderm differentiation. Cell nuclei were stained with DAPI (blue).
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
35
10/12/2010
4:38 PM
Page 35
Molecular Principles Underlying Pluripotency and Differentiation
How do we further differentiate definitive endoderm, mesoderm, or definitive ectoderm cells into the specific differentiated cell types that we are interested in? Due to space constraints, we will only focus on differentiation to three differentiated cell types of therapeutic interest — pancreatic β-cells and hepatocytes (definitive endoderm derivatives) and cardiomyocytes (a derivative of the mesoderm). In order to produce pancreatic β-cells and hepatocytes from ES cellderived definitive endoderm progenitors, we again rely on principles from developmental biology that inform us on how the nascent pancreas and liver are demarcated from within the definitive endoderm during development. Although the definitive endoderm is originally planar after its developmental formation, it is patterned into an anterior compartment (the anterior definitive endoderm) and a posterior compartment, and soon afterwards, the entire endodermal sheet involutes, forming a tubular structure known as the primitive gut tube.76 Correspondingly, the anterior aspect of the gut tube, the “foregut”, is generated from the anterior definitive endoderm — both the pancreas and the liver emerge from the foregut.75,107 However, whereas the liver arises from the anterior portion of the foregut (known as the “anterior foregut”),75,107 the majority of the pancreas arises from a slightly more posterior position within the foregut (known as the “posterior foregut”).75,79,107 How are the pancreatic and hepatic progenitors specified from within the foregut? The liver-producing anterior foregut is specified by several signals, including FGF1, FGF2, and BMP4,108,109 whereas the pancreatic anlagen is specifically specified by retinoid signaling110–112 and the inhibition of sonic hedgehog signals.75,113 Thus, after ES cells are specified to the definitive endoderm by Activin A and Wnt3a treatment, they can be directed to a hepatic fate by treatment with BMP4 and FGF2,81 or else they can be differentiated into pancreatic progenitors by treatment with retinoic acid, cyclopamine (a hedgehog signaling inhibitor), and FGF10 (which promotes the proliferation of pancreatic progenitors in vivo).19,79,114 The efficiency of pancreatic differentiation can be further increased by treatment with a chemical activator of PKC (Indolactam V).10 Thereafter, the ES-derived pancreatic progenitors spontaneously differentiate into pancreatic endocrine progenitors capable of giving rise to all endocrine cell types within the pancreas, including the insulin-producing β-cells.79 The β-cells and hepatocytes produced in vitro using these ES cell differentiation protocols are remarkably similar to their in vivo counterparts and
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
36
10/12/2010
4:38 PM
Page 36
K. M. Loh et al.
furthermore, they have been shown to have therapeutic utility to ameliorate disease in animal models. Human ES cell-derived β-cells endogenously express insulin (along with characteristic pancreatic transcription factors), they exhibit glucose-responsive and electrophysiologically-response insulin secretion, and they are ultrastructurally similar to their in vivo counterparts, as assayed by electron microscopy.79 Post-maturation, transplantation of these ES-derived β-cells can ameliorate the hyperglycemia of diabetic mouse models, thus validating their potential therapeutic utility.19 ES cellderived hepatocytes express genes that are characteristic of mature hepatocytes, store glycogen, and after transplantation, they can integrate into the damaged livers of mouse models.81 Thus, for cell replacement therapies targeting the pancreas and the liver, it appears that we have the relevant cell types at hand within the laboratory — the present challenges are more of the clinical and translational type (discussed below). Human ES cells can also be differentiated into cardiomyocytes, which are a mesodermal derivative. As aforementioned, the ES cell-derived mesendoderm can be differentiated into the mesoderm by a combination of Wnt3a, BMP4, and low concentrations of Activin A,97–99 which is consistent with in vivo developmental studies of gastrulation. However, after the mesoderm is established, Wnt signaling powerfully inhibits cardiac specification and instead specifies a hematopoietic or vascular fate.100,115–118 Consequently, treatment of ES cell-derived mesoderm cells with a Wnt antagonist (Dickkhopf-related protein 1; DKK1) elicits cardiac differentiation118 and specification of a Flk1-expressing and c-Kit-negative multipotent cardiac progenitor (Flk1low/c-Kit negative) that is capable of differentiating into all the major cardiac lineages — cardiomyocytes, vascular smooth muscle, and endothelium.82 The efficiency of cardiac specification may be enhanced by treatment with BMP4 and VEGF.82,118 These aforementioned ES-derived cardiac progenitors are capable of spontaneously differentiating into cardiomyocytes — these cardiomyocytes express characteristic marker genes such as cardiac troponin, and importantly, they are electrophysiologically active.82 Transplantation of human ES cell-derived cardiomyocytes into the infarcted hearts of murine models enhances cardiac function20 and upon transplantation, they can also pace pig hearts that are afflicted with complete heart block.119 Despite these promising results in differentiating human ES cells into a plethora of clinically-useful differentiated cell types, challenges still remain, as alluded to above. Here, we briefly discuss some of the issues that presently challenge the therapeutic employment of pluripotent stem cells.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
37
10/12/2010
4:38 PM
Page 37
Molecular Principles Underlying Pluripotency and Differentiation
For the sake of brevity, we review a few but not all of the current challenges — for further details, we refer the reader to a recent comprehensive review.5 Perhaps one of the most immediate problems is immunocompatibility — for any ES-based cell replacement therapy, if the transplanted ES-derived cells are not immunocompatible with the patient, they will evoke an immune response that will lead to their own destruction.55,120,121 Several innovative approaches have been proposed to overcome this immunocompatibility issue,120 but it still remains a grave concern. A recent brilliant strategy to circumvent this immunocompatibility issue would be simply to derive pluripotent stem cells that are isogenic to the intended patient — that is, to derive pluripotent stem cells from the actual patient that we intend to treat. As we have discussed before, it is possible through transcription factor overexpression to induce pluripotency in differentiated cells explanted from a patient, thus creating pluripotent stem cells (known as induced pluripotent stem (iPS) cells, as aforementioned) that are precisely isogenic and autologous to the patient.15–17 The creation of isogenic iPS cell lines from human patients will resolve the immunocompatibility challenge inherent to cell replacement therapies. However, the creation of patient-specific iPS cell lines for each patient is currently an intensive enterprise not well-suited for large-scale clinical deployment. Nevertheless, we envisage that in the future, certain technological innovations may increase the utility of this approach, thus allowing for the production of autologous patient-specific iPS cell lines for many patients. Another oft-overlooked challenge to ES-based cell replacement therapies lies in whether or not the transplanted cells will even be able to replace the physiological functions of the absent cells that they are intended to replace. While restoring near-normal numbers of the relevant cell type is one issue, certain cells (e.g. neurons) must be integrated into highly sophisticated cellular architectures and interface with other specific niche cells in order to prosecute their physiological function. Thus, it has been of interest to see whether or not disorganized aggregates of transplanted ESderived cells will be able to intuitively re-integrate themselves into their native architectures and thus resume the function of their in vivo counterparts.122 It has become apparent that simple transplantation of spatially-disorganized ES-derived cells may be insufficient to recapitulate the activity of the patient’s missing cells within certain organs such as the heart, where the organization of native cells into sophisticated tissue architectures engenders their physiological functions.123 Thus, for the
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
38
10/12/2010
4:38 PM
Page 38
K. M. Loh et al.
specific case of cardiomyocyte-based cell replacement therapies for the heart, it has been anticipated that any successful therapy would entail scaffolding the transplanted ES-derived cardiomyocytes into particular uniaxial conformations that will enable them to produce effective and productive muscular contraction. Transplanting ES-derived cardiomyocytes without any prior spatial organization of cells may indeed be of little therapeutic value, as unorganized masses of cardiomyocytes produce minimal effective net force when they contract.124 To this end, encouraging progress has been made with tissue engineering approaches to couple together cardiomyocytes into physiologically-productive architectures such that the cardiomyocytes will produce productive contraction after transplantation into the patient.124–126 Nevertheless, we note that there are still many complex cellular microarchitectures within the body whose structures may be incredibly difficult to replicate using tissue engineering approaches — such structures include the microvessels of endothelial cells which perfuse cardiac muscle.123 This has led to a suggestion that for the near future, it may be more productive to only develop ES-based cell replacement therapies that target organ systems such as the hematopoietic system where the concern of functionally integrating transplanting ES-derived cells into native tissue architectures may be less of an issue — in the specific case of the hematopoietic system, transplantation of any ES-derived hematopoietic cells into the bone marrow may not be subject to the concern of integration given the liquid nature of the bone marrow.121 We conclude that in the 12 short years since human ES cells were originally derived,39 extraordinary progress has been made in the campaign of differentiating human ES cells into clinically-relevant cell types. However, many formidable challenges on the translational and clinical side still remain — including the survival of the graft after transplantation20 amongst others. However, landmark victories have already been scored in using human ES cells to ameliorate severe conditions in animal models. Oligodendrocyte precursors attained from human ES cell differentiation have been shown to differentiate into oligodendrocytes upon transplantation into the injured spinal cord, and these cells have succeeded in enhancing axon remyelination and increasing locomotion of animals afflicted with spinal cord injury.61 Human ES-derived photoreceptor cells have been shown to integrate into retinal neural network circuitry following intraocular injection, and they have succeeded in regenerating some electrophysiological responses to light in blind mice.57 Human ES
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
39
10/12/2010
4:38 PM
Page 39
Molecular Principles Underlying Pluripotency and Differentiation
cell-derived pancreatic β-cells exhibit mature glucose-sensitive insulin secretion and have succeeded in suppressing the blood sugar levels of hyperglycemic diabetic mice.19 Thus, formidable challenges still remain, although significant key victories have been secured thus far. Now, we segue from the molecular principles that direct the differentiation of pluripotent stem cells to the molecular principles that underlie the condition of pluripotency — the very aspect of pluripotent stem cells that has made them so apt for therapeutic purposes.
Pluripotency Safeguarded by Transcription Factors — A Precarious Scenario Pluripotency is the ultimate freedom that can be attained by any mammalian cell2; the capability of pluripotent stem cells to generate any fetal cell type is unrivaled by any other cell type. The plasticity of pluripotent stem cells far surpasses the limited potency of multipotent adult stem cells within the adult body, which are capable of differentiating into only several lineages. What molecules impart pluripotent stem cells with their state of absolute freedom? What are the unique molecules or mechanisms that confer them with their unmatched plasticity? From a reductionist vista, the physiological function and phenotypic characteristics of any cell are largely determined by the proteins it expresses, which themselves are a function of a cell’s transcriptional and epigenetic programs.127 Thus, to understand the exclusive molecular agencies that confer pluripotent stem cells with their pluripotential character, we must turn to investigate the transcriptional and epigenetic networks that are operative within a pluripotent cell. A cell’s transcriptional output is a function of the summed activity of its transcription factors and epigenetic regulators — the net consensus of their functional dialogues either recruits or dispels the core transcriptional machinery (RNA polymerase and its cofactors) from transcribing a particular gene embedded within genomic DNA.128 The current dogma in the field holds that the role of epigenetic regulators is largely to govern the accessibility of transcription factors to their target sequences within chromatin and that it is transcription factors that directly effect the recruitment of the core transcriptional machinery to genes that are to be expressed.128–130
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
40
10/12/2010
4:38 PM
Page 40
K. M. Loh et al.
However, despite the notion that the gene expression patterns of a cell is dependent on contributions from both transcription factors and epigenetic regulators, thus far, most attention within the ES cell field has been directed towards the transcription factors that impart pluripotent stem cells with their pluripotent identity.131,132 Nevertheless, we feel it is important to elaborate on the critical roles that transcription factors play in the governance of cellular identities. Classic developmental studies have already ascertained that transcription factors are indispensible in the establishment and active maintenance of a cell’s lineage of choice. Striking examples include the finding that embryonic deletion of the transcription factor Pdx1 leads to the development of no pancreatic cell types whatsoever133 and the finding that expression of the transcription factor Sry within chromosomally female mice converts their gonads into testes.134 Recently, it was even found that deletion of Foxl2 within the adult ovary converts it into testes,135 affirming that transcription factors are required for actively safeguarding a cell’s lineage after its embryonic establishment. Unsurprisingly, transcription factors have also been accorded critical roles in the definition of the pluripotent character of ES cells. How can we determine whether a particular transcription factor is necessary for conferring ES cells with their pluripotentiality? Candidature is generally imparted by satisfaction of two criteria: the transcription factor must be expressed within ES cells and genetic ablation or suppression of the transcription factor must compromise the pluripotentiality of ES cells. Over the past decade or so, evidence has accrued that has highlighted three transcription factors that are at the heart of pluripotency — Oct4, Sox2, and Nanog.44,65,136,137 All three transcription factors are expressed within the pluripotent epiblast of the embryo and they are also expressed within ES cells: the explanted derivatives of the epiblast.44,65,137–140 Genetic deletion of Oct4, Sox2, or Nanog leads to compromise of the pluripotent epiblast, affirming that all three are functionally responsible for electing the epiblast to pluripotency.44,134,137 Although overwhelming attention has been paid thus far to this notorious “trinity” of pluripotency-enabling transcription factors,131,132 it has recently become clear that there are a plethora of other transcription factors that are also at the foundation of pluripotency — these additional transcription factors include c-Myc, Esrrb, Foxd3, Klf5, Ronin, Sall4, Tbx3, and Zfx.141–148 Here, we detail the molecular mechanisms by which we believe these transcription factors safeguard pluripotency and we also challenge existing
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
41
10/12/2010
4:38 PM
Page 41
Molecular Principles Underlying Pluripotency and Differentiation
models regarding the roles of these transcription factors in pluripotent cells. We begin by inspecting certain transcription factors critical for pluripotency and by summarizing simple phenomological observations regarding their roles in safeguarding pluripotency. Oct4 was the first transcription factor found to be indispensible for bestowing pluripotency upon cells. As aforementioned, it is ubiquitously expressed within the pluripotent epiblast138,139 as well as within mouse and human ES cells.140 Furthermore, its genetic deletion within the embryo leads to the inability to found the epiblast, and its genetic deletion within established ES cell lines leads to their immediate compromise.136 All these observations simply establish that Oct4 is critically required for the impartment of pluripotency, but they yield no insight into how Oct4 actually molecularly confers pluripotency upon stem cells. The first interesting observation into Oct4’s mechanism of action was that embryonic Oct4-/- cells fated to form the nascent epiblast instead form the trophectoderm136; this suggested that the function of Oct4 in safeguarding pluripotency is more or less to shield pluripotent cells from committing to the trophectoderm, thus encouraging them to remain within their undifferentiated, pluripotent state. This was confirmed by studies with actual mouse ES cells; suppression of Oct4 within mouse ES cells leads to their trophectodermal differentiation.149 Other studies with further transcription factors have revealed that differentiation is a common phenotype upon knockout or knockdown of cardinal pluripotency transcription factors within ES cells is differentiation. For example, genetic deletion or suppression of Sox2 within mouse ES cells leads into their differentiation into the trophectoderm141,150; knockdown of Esrrb elicits ectodermal and endodermal differentiation141; and knockdown of Tbx3 compels ectodermal differentiation.141 Thus, from this initial appraisal it appears that the general mechanism by which transcription factors safeguard pluripotency is to blockade ES cells from precociously differentiating into particular lineages, thus forcing them to remain in an undifferentiated and pluripotential state. Logically developing this argument, one might surmise that overexpression of these transcription factors would further consolidate the pluripotent character of ES cells by strongly repressing any precocious differentiation. Surprisingly, this notion is untrue in many circumstances. Overexpression of Oct4 not only does not reinforce the pluripotentiality of ES cells, but rather, it evokes their differentiation into the mesoderm and primitive endoderm.149 Likewise, overexpression
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
42
10/12/2010
4:38 PM
Page 42
K. M. Loh et al.
of Esrrb spurs endodermal differentiation141 and overexpression of Sox2 may provoke neuroectodermal differentiation.141 From this, we surmise that transcription factor kingship over pluripotency is inherently unstable. While deletion or suppression of specific transcription factors leads to the dissolution of pluripotency, overexpression of certain transcription factors leads to the downfall of pluripotency. For example, if we take the example of Sox2, it would appear that Sox2 might stabilize pluripotency by repressing trophectodermal differentiation,150 but instead, it appears that Sox2 also serves to subvert pluripotency, as when it is upregulated, it induces neuroectodermal differentiation.141,151 A similar example operates in the instance of Oct4, which at first assessment appears to safeguard pluripotency by blockading trophectodermal commitment136 — but instead, it seems that Oct4 also has interests contrary to pluripotency, as its overexpression elicits mesodermal and primitive endodermal differentiation.149 We propose that the alliance of transcription factors present within ES cells that supposedly safeguard pluripotency are a self-conflicted alliance; while each of them neutralizes differentiation to specific lineage(s), many (such as Oct4, Sox2, and Esrrb) indeed wish to compel ES differentiation into another specific lineage of interest. When all present in combination, these transcription factors counteract one another’s differentiation-inducing propensities and blockade differentiation into all possible lineages, and thus the net effect is (temporary) retention of pluripotency and the undifferentiated state. Selective downregulation of a certain transcription factor weakens its repression of the lineage(s) it represses, and other transcription factors that favor differentiation into that lineage become unopposed, thus driving differentiation into that lineage. Conversely, we propose that selective upregulation of a particular transcription factor leads it to overpower the other transcription factors that would otherwise repress differentiation into that lineage, thus commanding the ES cell to differentiate into the specific lineage that the upregulated transcription factor specifies. Our working model is attractive in that it concisely explains the potency of pluripotential cells to differentiate into all fetal cell types. The central question we hope to address in this section is what molecular mechanisms confer pluripotent cells with their extraordinary plasticity. We argue that their unmatched potency can be explained in part by the potent lineagedriving forces invoked by the transcription factors within them. It is curious to note that transcription factors critical for the development of
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
43
10/12/2010
4:38 PM
Page 43
Molecular Principles Underlying Pluripotency and Differentiation
specific tissues, such as neural tissues (Sox2 and Foxd3)137,152 and epithelial tissues (Klf4),153 are expressed within ES cells — it is interesting that these tissue-specific transcription factors are “re-purposed” from their tissue-specific roles and that they are expressed within ES cells. Given this observation, we propose that the pluripotentiality of ES cells can be partially explained by the expression of transcription factors that drive differentiation into many major lineages; thus, ES cells have a representation of all the transcription factors required for their differentiation into the major lineages that the fetus might require. The pluripotency of ES cells is constructively expended during differentiation by upregulation of transcription factors that selectively favor differentiation to a certain lineage (e.g. mesoderm) and the concomitant downregulation of transcription factors that would otherwise drive differentiation to alternative lineages. By downregulation of factors required for differentiation into these alternative lineages, these ES cells lose their competency to differentiate into any lineages (e.g. downregulation of Sox2 might make neural differentiation inaccessible), thus forcing ES cells to differentiate into the lineage favored by the current set of upregulated transcription factors. Thus, in such a model, it would appear that a precise level of expression of each particular transcription factor is critical for the long-term insurance of pluripotency within ES cells; for example, either upregulation or downregulation of transcription factors such as Oct4 or Esrrb would be fatally compromising to pluripotency. We note at this point that while some transcription factors appear to bias ES cells towards differentiation into particular lineages (such as the aforementioned examples of Oct4, Sox2, and Esrrb), this is not likely the situation for all transcription factors — in fact, overexpression of Nanog or Zfx within ES cells renders them especially recalcitrant against differentiation.65,146
Pluripotency is the State of Exception Altogether, we conclude that the transcription factors that safeguard pluripotency indeed comprise a self-conflicted coalition, with many members wishing to drive differentiation to their own lineage of interest; thus, the very benefactors of pluripotency wish to abolish it upon their own upregulation. We believe that this conveniently explains the innate transiency of embryonic pluripotency during development — pluripotential cells of the epiblast are established by E4.5 within the post-implantation
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
44
10/12/2010
4:38 PM
Page 44
K. M. Loh et al.
blastocyst,3 yet they lose their pluripotency shortly thereafter by E6.7.25 Clearly, embryonic pluripotency is only intended to be transient; in fact, obviously persistence of pluripotent cells in the embryo past the proper time would be extraordinarily deleterious — if there is co-existence of pluripotent cells alongside fetal cells, these pluripotent cells would eventually and spontaneously differentiate into masses of disorganized cells. In fact, the etiology of naturally-occurring teratomas and teratocarcinomas within human patients lies in the incorrect persistence of pluripotent cells past early embryonic development, after which they differentiate and either form benign or aggressive cancers, teratomas or teratocarcinomas, respectively.154,155 Thus, once epiblast cells are formed, their pluripotentiality must be rapidly exploited to generate the various fetal tissues, and afterwards, their pluripotent character should be lost. From this, we surmise that pluripotency is the state of exception — not the state of constancy. In fact, the derivation of stable, permanentlypluripotent ES cell lines from the epiblast represents the immortalization of pluripotency, which is developmentally illegitimate. Thinking in this manner, the pluripotency of ES cells, borrowed from the pluripotency of the epiblast, should also be unstable. We note that this is well-explained by our model above regarding the self-conflicted transcription factor alliance that watches over pluripotency; slight downregulation or upregulation of a particular transcription factor could lead to the collapse of pluripotency. Thus, we propose that the inherent transience and instability of embryonic pluripotency and that of ES cells may even be attributable to transcriptional noise that leads to small changes in the expression levels of critical pluripotency transcription factors, which translate into differentiation into specific lineages. Nevertheless, molecular arguments have been made to the effect that the pluripotent state of ES cells is self-renewing and stable. For example, it has been shown that the Oct4, Sox2, and Nanog transcription factors bind one another’s promoters and upregulate each other’s expression,156–160 and this has been thought to comprise a self-reinforcing and recursive circuit that sustains high levels of expression of all three transcription factors, thus safeguarding pluripotency (Fig. 3). We surmise that this autoregulation, which should continually enforce high levels of expression of Oct4, Sox2, and Nanog, explains how pluripotency could even be maintained in the first place — through the constant re-stimulation of expression of these genes to ensure that they repress the differentiation urges of one another and other ES-expressed transcription factors.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
45
10/12/2010
4:38 PM
Page 45
Molecular Principles Underlying Pluripotency and Differentiation
Figure 3. A propensity for differentiation is inherent to the core transcription factor circuits underlying pluripotency. The core transcriptional circuit operative within mouse ES cells is presumed to be commanded by the transcription factors Oct4, Sox2, and Nanog (left). Strikingly, these transcription factors not only upregulate self-renewal genes, but they also upregulate differentiation genes. Oct4, Sox2, and Nanog bind each other ’s promoters and upregulate one another (top), thus ensuring high expression levels of all three transcription factors and ensuring continuance of the circuit. However, Oct4, Sox2, and Nanog also bind the Tcf3 promoter and Oct4 and Sox2 also bind the Fgf4 promoter (bottom), upregulating expression of these two differentiation genes. The Tcf3 transcription factor represses Oct4, Sox2, and Nanog transcription, while FGF4 is an autocrine/paracrine growth factor that signals for ES cell differentiation. Given that core ES pluripotency transcription factors upregulate these potent differentiation factors, we surmise that a proclivity to differentiate is molecularly inherent to the Oct4-Sox2-Nanog transcriptional circuit — these three transcription factors temporarily safeguard pluripotency when they are expressed at high levels but they also upregulate FGF4 and Tcf3, thus keeping ES cells poised to differentiate even as they self-renew.
However, it should be mentioned that Oct4 and Sox2 not only bind each other’s promoters but they also bind the Fgf4 promoter and upregulate Fgf4 expression161–163 — FGF4 secreted by ES cells serves as an autocrine/paracrine signal that drives neural and mesodermal differentiation through MAPK signaling.164 Thus, interestingly, while this Oct4-Sox2 selfactivating circuit presumably acts to safeguard pluripotency by upregulating levels of their own expression, it also induces the expression
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
46
10/12/2010
4:38 PM
Page 46
K. M. Loh et al.
of FGF4, a potent differentiation signal. We surmise that these recursive circuits between ES-expressed transcription factors does not simply ensure pluripotency but rather keeps ES cells poised to differentiate by simultaneously enacting expression of the critical differentiation signals, such as FGF4. Furthermore, we note that Oct4 and Nanog bind the promoter of Tcf3 and are believed to activate its expression165 — the Tcf3 transcription factor in turn represses Oct4, Sox2, and Nanog by binding to their promoters, thus compelling ES cell differentiation.166–168 This recursive activation of a differentiation signal (FGF4) and a differentiation-inducing transcription factor (Tcf3) by these ES cell transcriptional circuits further provides evidence for the notion that transience and a proclivity to differentiate is molecularly inherent to the pluripotency of ES cells (Fig. 3).
Many Roads Lead to Pluripotency As aforementioned, genome-scale transcriptional profiling44,169–171 and genome-scale transcription factor binding-site mapping160,172–175 within human and mouse ES cells have given evidence that Oct4, Sox2, and Nanog comprise a “trinity” of transcription factors essential for safeguarding pluripotency. Thus, perhaps it came as a surprise that Nanog was not found to be expressed by all ES cells — within a culture of mouse ES cells, it appears that only 50–80% of cells express Nanog,176,177 which was thought to be indispensible for the pluripotency of both the epiblast and ES cells.44,65 Then what is the status of the remaining 20–50% of cells within ES cell cultures that do not express Nanog protein? Are they even pluripotent? Are half of all cells within steady-state ES cell cultures simply differentiated or nullipotent? Strikingly, the Nanog-negative ES cells are indeed authentically pluripotent and are not differentiated, as they are capable of self-renewal and they express SSEA1,177 which is diagnostic of pluripotent stem cells.178 Amazingly, ES cells still retain pluripotency even after complete genetic ablation of Nanog, as Nanog-/- ES cells are capable of efficiently contributing to chimeras after complementation with diploid blastocysts.177 Furthermore, Nanog-negative cells can re-express Nanog again, thus becoming Nanog positive, and these Nanog positive cells can downregulate Nanog, becoming Nanog negative and generating the Nanog negative
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
47
10/12/2010
4:38 PM
Page 47
Molecular Principles Underlying Pluripotency and Differentiation
subpopulation within the culture.177 Juxtaposition of this observation with the previous observations that Nanog is unequivocally indispensable for conferring pluripotency to the epiblast44,179 presents a contradiction of sorts. To this end, a beautiful model has been constructed to account for the apparently divergent function of Nanog within the pluripotent epiblast and in ES cells. This model premises that Nanog is indispensable a for the construction of pluripotency — that is, the initial conferment of pluripotency to inner mass cells to construct the epiblast — but that afterwards, Nanog becomes dispensable for the active maintenance of pluripotency within established pluripotent cells, such as ES cells.177 This hypothesis that Nanog functions in the construction but not the maintenance of pluripotency has been substantiated by provocative findings that examine the construction of pluripotency within differentiated cells179 (discussed below). Thus, it can be concluded that creating pluripotency and safeguarding pluripotency are molecularly distinct processes that have different molecular requirements. A more tangible analogy might be that constructing a structure and maintaining it have dramatically different demands and requisite skills. It should be emphasized that Nanog appears to be relatively unique in this capacity. For example, genetic deletion of either Oct4136 or Ronin147 within ES cells leads to their immediate compromise. Thus, Oct4 and Ronin are indispensable not only in the construction of pluripotency within the epiblast, but also in the active maintenance of pluripotency once it has been established.136,147 This explains why Oct4 expression is invariant and that it is homogeneously expressed within all ES cells in cell culture — any perturbation in its levels would lead to the collapse of pluripotency.49 Nevertheless, it should be noted that Nanog-negative ES cells or Nanog-/- ES cells have an altered morphology, decreased selfrenewal capacity, and they highly vulnerable to differentiation (although they still pluripotent, as per their capacity to complement diploid blastocysts).177 However, subsequent studies have suggested that Nanog may not be alone in the class of apparently “dispensable” pluripotency transcription factors. The pluripotency transcription factors Klf4, Rex1, Stella, and Tbx3 have also recently been found to be variably expressed within ES cells.176,180,181 It has been found that ∼20–25% of ES cells within a culture do not express Klf4 or Rex1176 and that 70–80% of ES cells do not express
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
48
10/12/2010
4:38 PM
Page 48
K. M. Loh et al.
Stella or Tbx3 — thus, it is a minority of ES cells that actually express Stella or Tbx3.176,181 The fluctuation in expression levels of these transcription factors have often-underappreciated functional consequences. Although Rex1-negative and Stella-negative ES cells express similar levels of Oct4 as their Rex1positive and Stella-positive counterparts,180,181 they express higher levels of differentiation markers such as Brachyury (mesendoderm), Fgf5 (postimplantation epiblast182), and Sox17 (endoderm)180,181 (summarized in Figs. 4e–4g). On the contrary, Rex1-positive ES cells have upregulated the expression of both Nanog and Stella and they have also downregulated expression of the above differentiation markers.180 Taken altogether, this suggests that individual ES cells are not transcriptionally nor functionally equivalent to one another, as previously assumed.169,170 Although there is more work to be done, this suggests that there are at least discrete compartments within ES cell cultures: a “primitive” compartment demarcated by Nanog, Rex1, and/or Stella expression that has enhanced self-renewal capacity and is refractory to differentiation; and is a “differentiation-poised” compartment defined by ES cells that have downregulated Nanog, Rex1, and/or Stella expression that have elevated expression of differentiation genes. It appears that cells readily transit from one compartment to the other stochastically — cells that do not express Nanog can re-express Nanog177 and cells that do not express Rex1 or Stella can re-express these two genes.180,181 The implications of this are discussed in more detail elsewhere.132,183 Nevertheless, the intent of this discourse is to support our hypothesis that multiple, discrete transcriptional programs are operative within ES cells (Fig. 4), as it has been assumed previously that all individual ES cells share the same transcriptional program.170 Given that Nanog-/- ES cells are still genuinely pluripotent,177 we speculate that there are at least two distinct transcriptional programs capable of producing a pluripotent cell — one that exists independent of and is unreliant on Nanog, and another that is supported by Nanog. The existence of a pluripotency network that exists in the absence of Nanog suggests that it is not a pivotal foundation of the pluripotent transcriptional network, as are Oct4 and Ronin, whose constant expression is imperative to retain pluripotency. Thus, what are the molecular differences between the two networks produced when these accessory factors are present or absent? For example, what specific genes are upregulated or downregulated by Nanog overexpression that imparts Nanog-positive ES cells with their
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
49
10/12/2010
4:38 PM
Page 49
Molecular Principles Underlying Pluripotency and Differentiation
Figure 4. Distinct transcriptional programs operative within mouse ES cells. An illustration of multiple and distinct transcriptional programs that are operative within mouse ES cells, defined by the presence of absence of particular pluripotency transcription factors (Oct4, Sox2, Stella, Rex1, and Nanog are examined here). A differentiation marker, Fgf5, is also examined. The red arrows throughout the figure highlight which transcription factor is being decreased from its initially high level of expression within condition “B” (left). The blue-shaded diminuendos (bottom) also highlight which transcription factor is being decreased in the specific program being examined. Note throughout that irrespective of whatever other transcription factors are being downregulated, Oct4 expression is invariant throughout in all programs. This figure was based on the authors’ interpretation of data presented within several references.177,180,181 (A) Figure legend: strong color means high levels of expression, moderate color means medium levels of expression, and no color means little to no expression; blue boxes denote pluripotency transcription factors, while red boxes represent levels of the differentiation marker Fgf5. (B) The “ideal” ES cell previously assumed by previous publications, which expresses all five pluripotency transcription factors under examination at high levels. (C) and (D) ES cell transcriptional programs that are unreliant on Nanog. Decreased levels or complete absence of Nanog have no notable impact on the expression levels of the other four pluripotency transcription factors and do not upregulate Fgf5. Thus, Nanog is largely dispensable for upregulating other transcription factors and does not exert a repressive effect on the differentiation program demarcated by Fgf5 expression. (E) An ES cell transcriptional program unreliant on Rex1. However, absence of Rex1 leads to decreased expression of Stella and Nanog and upregulation of Fgf5. Thus, Rex1 regulates Stella and Nanog expression and represses Fgf5. (F) An ES cell transcriptional program partially unreliant on Stella. Downregulation of Stella expression from its original high levels of expression within condition “B” leads to downregulation of Sox2, Stella, and Nanog, but does not markedly upregulate Fgf5. (G) An ES cell transcriptional program completely unreliant on Stella. Total absence of Stella also leads to severe downregulation of Rex1 and moderate downregulation of Sox2 and Nanog, concomitant with upregulation of Fgf5. Thus, it can be surmised that Stella regulates more pluripotency factors than does Rex1 (it regulates Sox2 and is critically dependent for Rex1 expression), and that it also serves to repress Fgf5.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
50
10/12/2010
4:38 PM
Page 50
K. M. Loh et al.
differentiation-refractory phenotype? This question warrants further investigation. The converse inquiry — what gene expression changes are produced within to the pluripotency network upon Nanog downregulation or deletion — is also an interesting question. For example, Nanog has been directly shown to activate transcription from the Rex1 promoter and upregulate Rex1 expression,184 and various genome-wide transcription factor binding site-mapping studies (discussed below) have predicted that Nanog regulates the expression of many genes necessary for maintaining pluripotency.172,174,175 In fact, the most recent of such studies alleges that Nanog may bind nearly 1300 gene promoters within mouse ES cells175; it appears that Nanog also binds the Oct4 and Sox2 promoters.172 How can ES cells withstand the depletion of Nanog if it is presumably required for appropriate expression of all these genes? It has been suggested that Nanog is indeed not required for the appropriate regulation of many of these genes; indeed, Nanog-/- cells express comparable levels of Oct4, Sox2, Rex1, Stella, Tcl1, and many other pluripotency genes as compared with wild-type ES cells.177 We conclude that Nanog seems to be an optional accessory to the pluripotency transcriptional network, affording additional protection against stochastic differentiation but still ultimately dispensable. Accordingly, overexpression of Nanog safeguards pluripotency even in conditions when differentiation is normally elicited — Nanog overexpression enables mouse ES cell self-renewal and continued pluripotency even when both LIF and BMP4 are withdrawn65,185 and Nanog overexpression also permits for human ES cell growth in the absence of feeders.186 Nevertheless, further transcriptional studies are required to identify the ancillary genes whose expressions are dysregulated when Nanog is absent — presumably, appropriate expression of these genes within Nanogpositive ES cells confers them with their resistance to differentiation. Given that Klf4, Rex1, Stella, and Tbx3 expressions are also variable within ES cells (with the majority of ES cells at any given time not even expressing Stella nor Tbx3), we similarly surmise that these transcription factors may represent additional optional accessories, affording enhanced self-renewal capacity and resistance against differentiation but nonetheless dispensable. Thus, it appears that there are a multitude of transcriptional programs operative within mouse ES cells which differ in the expression of multiple transcription factors such as Nanog, Rex1, and Stella (Fig. 4) — each of these transcriptional programs imparts a different phenotype, such as enhanced self-renewal or else enhanced proclivity for differentiation.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
51
10/12/2010
4:38 PM
Page 51
Molecular Principles Underlying Pluripotency and Differentiation
We also have recently suggested that there are indeed many transcriptional programs that can produce discrete types of pluripotent cells41 — for example, mouse ES cells, epiblast stem cells (EpiSCs),32 and embryonic germ (EG) cells, which are all pluripotent and are capable of multilineage differentiation, and are all transcriptionally distinct and different from one another. 32,33,187 The transcriptional program operative within each subclass of pluripotent cell type differs on the in vivo origin of the pluripotent cell, whether or not it is explanted from the early post-implantation epiblast (ES cells), the mature post-implantation epiblast (EpiSCs), or derived from primordial germ cells (EG cells). From these above observations, we conclude that there are many transcriptional roads that lead to pluripotency.
Installing the Epigenetic Programs of Pluripotency Our discussion on pluripotency thus far has been solely focused on a cadre of transcription factors. Nevertheless, as we mentioned at the beginning of this chapter, gene expression is a function of the combined activities of transcription factors and epigenetic regulators within the cell — it is the joint consensus of both transcription factors and epigenetic regulators that determines whether or not a gene is expressed or repressed.128–130 Transcription factors do not operate unilaterally — transcription takes place in the context of chromatin, and thus, we premise that a functional negotiation between transcription factors and epigenetic regulators is critical to ensure proper patterns of gene expression within pluripotent cells. Despite this, there is an emerging opinion within the field that pluripotency is almost exclusively constructed and maintained by transcription factors and that epigenetic regulators play a minimal role in safeguarding pluripotency.132 Here, we address the role of epigenetic programs within ES cells and we seek to provide a more complete interpretation of the present situation. As discussed above, we premise that transcription factors associated with differentiation into major lineages are expressed (albeit at low levels) within ES cells, and it is this expression of the transcription factors that imparts ES cells with their pluripotential character — the expression of these transcription factors makes ES cells transcriptionally competent to differentiate into these lineages. For example, ES cells do
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
52
10/12/2010
4:38 PM
Page 52
K. M. Loh et al.
express key differentiation genes such as Brachyury.177 If the ES cells have access to these differentiation/developmental programs and a number of these differentiation programs are operative within ES cells at low levels (i.e. the mesendodermal program orchestrated by Brachyury), one might premise that ES cells might express many different genes (corresponding to many possible downstream lineages) as compared with differentiated cells, which would only express genes that are characteristic to their single, fixated lineage. Indeed, while certain differentiated cells express only 10–20% of genes within the genome, it appears that ES cells express 30–60% of genes encoded in the genome188 — which is a surprisingly high proportion. This evidence for pervasive genome-wide transcription within ES cells189 argues that there may be unique epigenetic features within ES cells that enable such pervasive transcription, which is not observed in differentiated cells. Global observations suggest that there is a far lower content of repressive heterochromatin within ES cells as compared with differentiated cells,190 thus perhaps explaining partially the phenomenon of pervasive transcription. Evidence for a globally permissive chromatin state has also been reinforced by recent findings that there is global histone acetylation within ES cells191 and the observation that ES cells have a 10fold less repressive H3K9 dimethylation as compared with certain differentiated cell types.192 Furthermore, it appears that various histone components are more loosely assembled on the ES genome than on the genomes of differentiated cells.190 We speculate that this would generally help facilitate transcription on a global scale if nucleosomes or their accessories are easier to disassemble within ES cells. These results are suggestive of a globally open epigenetic state within ES cells that permits for preview of many possible lineage programs — selection and commitment to a particular differentiation program would be anticipated to induce epigenetic closure of the alternative lineage programs. Indeed, heterochromatin foci become much more frequent and larger within differentiated cells.190 There is also an indispensable role for ATP-dependent chromatin remodelers in establishing and safeguarding pluripotency — such chromatin remodelers have been canonically found to disrupt nucleosomes, thus facilitating transcription.193,194 Indeed, embryonic genetic deletion of the chromatin remodelers Brg1, Baf47, Baf155, or Snf2h leads to periimplantation lethality,195–198 which is indicative of failure of proper specification or operation of the pluripotent epiblast.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
53
10/12/2010
4:38 PM
Page 53
Molecular Principles Underlying Pluripotency and Differentiation
Interestingly, knockdown of the chromatin remodeler Chd1 within ES cells abrogates their capacity for multilineage differentiation and elicits heterochromatinization of euchromatin199 — this affirms that a globally transcriptionally-permissive chromatin state allows for preview of multiple lineage programs and that blinding this preview by global heterochromatinization compromises any possible lineage commitment. This is paralleled with studies on the chromatin remodeler Baf250a; Baf250a-/embryos failure to specify the mesoderm during gastrulation,200 suggesting that the particular function of Baf250a within the pluripotent epiblast is to keep mesodermal-specific genes “open” and accessible for transcriptional activation. Nevertheless, the role of epigenetic repression in ES cells should not be discounted either; while we believe ES cells operate within a globally transcriptionally-permissive chromatin state (thus accounting for the phenomenon of pervasive transcription), obviously high levels of expression of differentiation genes would elicit differentiation, thus collapsing the undifferentiated state. Findings that the DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b are dispensable for ES cell self-renewal201 have led to the assertion that maintenance of pluripotency is unreliant on epigenetic repression.132 We do not find it likely that this assertion is correct. Hypomethylated single-knockout Dnmt1-/- ES cells or double-knockout Dnmt3a-/- × Dnmt3b-/- ES cells are unable to efficiently prosecute terminal differentiation,202 thus invalidating their pluripotency. Furthermore, both Oct4 and Nanog directly associate with transcriptional repressor complexes that contain factors such as histone deacetylases (Hdac1 and Hdac2) and the H3K4 histone demethylase Lsd1.203–205 This suggests that epigenetic repression is an obligatory feature of the pluripotent state. Altogether, we conclude that functional negotiation between transcription factors and epigenetic regulators is necessary to safeguard pluripotency. It appears that neither class of factors is dispensable to preserve pluripotency requiring a functional cooperativity between the two to instate pluripotency. A globally open chromatin state is required to allow pluripotent cells to preview possible differentiation programs, thus making them competent to access these programs. However, a level of epigenetic repression is still requisite to ensure that expression of these differentiation genes remains at low levels such that they do not overwhelm the undifferentiated state of pluripotent cells. Furthermore, it is likely that the role of chromatin remodelers in conferring a globally open chromatin state is a unique feature inherent to ES cells (or to stem cell populations in
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
54
10/12/2010
4:38 PM
Page 54
K. M. Loh et al.
general), and that these chromatin remodelers would be unnecessary for differentiated cells which are already fixated within their lineage of choice — this is supported by the observation that Brg1 is dispensable within differentiated cells such as fibroblasts.206
Systemic Architectures Confer Pluripotency Above, we have approached pluripotency from a relatively minimalist “single gene” approach by discussing the effects of single genes. It is becoming increasingly apparent that these pluripotency-enforcing transcription factors do not simply work on their own to enact pluripotency. While disruption of individual key genes is sufficient to compromise pluripotency, it appears that pluripotency is actually constructed at a systems level from extensive collaboration between these transcription factors and their cofactors (epigenetic regulators and other factors). Thus, we premise there is a high-level architecture to the transcriptionalepigenetic network that underlies pluripotency. Perhaps a relatively simple example is the finding that the Oct4 and Sox2 transcription factors cooperatively bind DNA together, physically forming a heterodimer that regulates transcriptional activity.163 This Oct4/Sox2 heterodimer binds a particular motif, named the “Oct/Sox motif,” within gene regulatory elements, and it is through binding to this motif that the Oct4/Sox2 heterodimer governs the transcription of a variety of genes, including Fbx15, Fgf4, Nanog, Osteopontin, Utf1, and themselves (Oct4 and Sox2).156–159,161–163,207–210 Efficient binding of Oct4 to certain promoters is critically dependent on the presence of Sox2,211 and activation of the Fgf4 promoter requires the presence of both Oct4 and Sox2161; either transcription factor by itself is unable to stimulate transcription. Surprisingly, although Oct4 itself binds to the Osteopontin promoter and activates Osteopontin transcription, the presence of Sox2 and the formation of the Oct4/Sox2 heterodimer ultimately leads to Osteopontin repression208 — thus, this is a clear instance where cooperativity between both Oct4 and Sox2 alters gene expression patterns. These examples illustrate that in some instances, individual factors are inactive and that it is the cooperation between multiple factors that enacts novel transcriptional activities; this helps explain emergent properties at the systems-level within ES cells that are not present at lower levels when individual factors are examined.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
55
10/12/2010
4:38 PM
Page 55
Molecular Principles Underlying Pluripotency and Differentiation
Beyond these limited examples concerning only Oct4 and Sox2, it is clear that recruitment of cofactors by transcription factors is generally required for them to prosecute their transcriptional activities. Recent immunoprecipitation experiments followed by mass spectrometry have identified the direct protein interactants of Oct4, Nanog, and other transcription factors related to pluripotency.204,205,212 In addition to Sox2, Oct4 interacts with a large variety of transcription factors, such as Esrrb, Klf4, Klf5, Nanog, and Sall4, all of which have been shown to be responsible for safeguarding pluripotency.203,205 Given that heterodimerization of Oct4 and Sox2 is necessary for correct expression of Fgf4 (and that either of the two that either of transcription factors alone are insufficient161), it is tempting to speculate that Oct4’s interactions with these additional pluripotency transcription factors may produce novel transcription factor heterocomplexes which possess unique transcriptional activities. Besides transcription factors, Oct4 also interacts directly with a surprisingly diverse plethora of proteins that are associated with a number of complexes, such as the SWI/SNF chromatin remodeling complex (Brg1 and Baf155),203,205 the NuRD transcriptional repressor complex (Chd4, Hdac1, Mbd3, and others),203,205 a histone chaperone complex (Hira and others),203 and the polycomb complex PRC1 (Phc1 and others)205; Oct4 even associates with DNA repair enzymes, helicases, DNA replication factors, and histones.203 Nanog has also been shown to interact directly with a number of similar factors, including various transcription factors (Sall4 and others),204,212 the REST transcriptional repressor,212 and factors within the NuRD and Sin3a-HDAC transcriptional repressor complexes.204,212 Although such mass spectrometry methods have some inherent error and results from such studies require further validation,131,213 such results raise interesting possibilities that may help explain how pluripotency is enacted by a multitude of factors in combination — the direct interaction of these proteins might impart system-level functions that the individual proteins themselves may not possess, creating novel transcriptional activities and allowing these complexes to exert specific regulatory effects on different genes. For example, it is interesting to speculate that when Nanog functions as a homodimer with itself 214 that it may target different genes than it would if it is assembled as a heterodimer with Sall4 or another transcription factor. Nevertheless, such speculation certainly requires functional validation. Another interesting contingency raised by the above results is that transcription factors such as Oct4 might co-target different cofactors to specific genes in order to either activate or repress them: for
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
56
10/12/2010
4:38 PM
Page 56
K. M. Loh et al.
example, Oct4 might repress a differentiation gene by recruiting the NuRD repressive complex or the polycomb repressive complex PRC1, or else it might selectively activate transcription of another gene by recruiting SWI/SNF chromatin remodelers or other co-activators. Finally, another interesting facet of ES cells that suggests inherent higher-level molecular architecture in pluripotency is that many genes within ES cells may be potentially co-regulated by multiple transcription factors. Recent studies to map the binding sites of pluripotency transcription factors within the ES cell genome have revealed that Oct4, Sox2, Nanog, and other pluripotency transcription factors co-target many of the same genes — for example, it has been estimated that Oct4 and Nanog bind 44.5% of the same genes within mouse ES cells.160,172,174,175 Furthermore, when pluripotency transcription factors co-bind the same gene within ES cells, the binding sites for these transcription factors are mapped to be extremely close together, suggesting that these transcription factors may be assembling into a complex that is binding the same regulatory element within a given promoter or enhancer region.175 This conjecture has been further fueled by recent genome-wide mapping of binding sites for nine pluripotency transcription factors (Oct4, Sox2, Klf4, Nanog, Dax1, Rex1, Nac1, Zfp281, and c-Myc), which revealed that more than 100 promoters are co-bound by seven of the above nine factors, suggesting the assembly of massive multi-factor complexes at certain regulatory regions.175 This above observation has led to the development of the “enhanceosome” model whereby certain promoters are bound by and coordinately governed by a ponderous ensemble of transcription factors; it is interesting to note that genes predicted to be bound by these hypothetical ES cell enhanceosomes are preferentially expressed, whereas genes bound by single pluripotency transcription factors are preferentially repressed in ES cells and become activated during differentiation.174,175 This again has led to further speculation that promoter activity within ES cells requires co-binding by multiple transcription factors (the hypothetical “enhanceosome”), whereas when transcription factors bind alone to a gene, they either function as repressors or else their activating function is insufficient and requires potentiation by additional transcription factors. While such speculation is certainly interesting, it is imperative to note that to date, no physical “enhanceosome” complex has actually been physically purified from ES cells where several transcription factors have been found to exist co-bound to one another at a single given moment. Thus,
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
57
10/12/2010
4:38 PM
Page 57
Molecular Principles Underlying Pluripotency and Differentiation
despite the extremely close co-localization of transcription factor binding sites upon certain promoters,175 some caution should be taken regarding this matter. Furthermore, one should be aware that there are technical issues inherent to such chromatin immunoprecipitation (chIP) studies131 and that transcription factor binding to a promoter does not necessarily mean that the transcription factor controls the transcription of its associated gene. Nevertheless, should such an ES cell enhanceosome exist, it would be interesting to see if it recruits any specific co-activators that prosecute its preferential upregulation of the genes that it targets. We conclude that individual pluripotency factors do not operate individually to confer pluripotency upon stem cells. Instead, it appears that systems-level communication between pluripotency factors, in the form of direct interactions and the assembly of physical complexes (such as the Oct4/Sox2 heterodimer or hypothetical multi-transcription factor enhanceosomes) or else co-regulation of common target genes by multiple transcription factors — is what actually enacts pluripotency. Single factors are insufficient to construct pluripotency — collaboration amongst a plethora of transcription factors and epigenetic regulators is what molecularly confers pluripotency.
Recreating Pluripotency As we have stated above, a cell’s lineage is directly defined by its gene expression pattern127,215; it is its characteristic gene expression pattern that confers a pluripotent stem cell with its pluripotent status, and likewise, it is a unique gene expression pattern that defines the phenotypic characteristics of a cell such as a neuron. Interestingly, gene expression is highly malleable and is directly subject to continual supervision, as transcription is regulated by the transcription factors and epigenetic regulators present within the nucleus at any given moment.216 These two observations — that gene expression patterns define cellular lineages and that gene expression is highly plastic and is continually determined by the transcription factors and epigenetic regulators (henceforth, “nuclear regulators”) — raise an interesting question. Are cellular lineages “reprogrammable”? If a cell’s lineage is constantly defined by the presence of that particular lineage’s nuclear regulators within a nucleus, would its lineage immediately change to another one if its present nuclear regulators were substituted by those of another lineage?
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
58
10/12/2010
4:38 PM
Page 58
K. M. Loh et al.
It would seem so. Taking development as an example, it is obvious that cellular lineages are reprogrammable to an extent. Cellular differentiation during development (such as pancreatic differentiation) is simply the iterative replacement of the epiblast’s nuclear regulators with those of the definitive endoderm’s, followed by replacement of the endoderm’s nuclear regulators with those of pancreatic progenitors, and so on and so forth. Whilst this seems logical in theory, it has long been held that lineage reprogramming is indeed not always possible; while lineage reprogramming from a progenitor cell to a more differentiated cell is a developmentally “legitimate” option (as per the aforementioned example of pancreatic development), the reverse — “de-differentiation” of a differentiated cell into a progenitor cell type — has been assumed to be impossible. There is an immediately evident physiological rationale for why this should be true; it would obviously be catastrophic if fetal development were reversible. Thus, it has been long-held that embryonic development is a unidirectional process and that the earliest step of fetal development — the differentiation of the pluripotent epiblast into the major fetal lineages — was thought to be especially irreversible. Consistent with this notion, there are indeed powerful molecular mechanisms that exist to ensure that pluripotency is permanently extinguished during the differentiation of pluripotent cells and that pluripotency can never be reacquired by the epiblast’s differentiated progeny. Such developmental “safeguard” mechanisms include a specialized arm of epigenetic repression orchestrated by the G9a histone methyltransferase and DNA methyltransferases; these two classes of epigenetic repressors recruit each other during ES cell differentiation to transiently silence the Oct4 and Nanog promoters, and then to enact stably DNA methylation of these promoters, permanently ensuring that Oct4 and Nanog cannot be reexpressed within differentiated cells. We surmise that this is the molecular explanation for why not even multipotent stem cells such as hematopoietic stem cells and neural stem cells express Oct4.170 Thus, it was due to the presence of this specialized safeguard and others like it that it was thought that “de-differentiation” of a differentiated cell into a pluripotent epiblast state was especially forbidden. It was a pivotal decisive study by Takahashi and Yamanaka that provided unequivocal evidence that pluripotency indeed could be recreated within differentiated cells.47 These authors showed that overexpression of Oct4, Sox2, Klf4, and c-Myc is sufficient to recreate pluripotency within non-pluripotent cells — thus directly converting differentiated cells (such
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
59
10/12/2010
4:38 PM
Page 59
Molecular Principles Underlying Pluripotency and Differentiation
as fibroblasts) into pluripotent stem cells.47 within ES cells, transcription factors control the expression of genes that define the pluripotent state, as aforementioned. Introduction of these key nodes of the pluripotency network reactivates the effectors of the pluripotent condition, thus effectively recreating pluripotency in differentiated cells. Subsequent studies by Yamanaka and other members of the field have expanded on this, decisively demonstrating that a plethora of differentiated cell types,7 such as fibroblasts, keratinocytes,217 and hematopoietic cells,218 can be reprogrammed into pluripotent stem cells by the overexpression of Oct4, Sox2, Klf4, and c-Myc. It is evident that the introduction of these transcription factors alone is nearly entirely sufficient to reconstruct the molecular architecture underlying pluripotency, as the pluripotent stem cells produced by the overexpression of these transcription factors, known as “induced pluripotent stem cells” (iPS cells), are extremely similar to bona fide ES cells in terms of gene expression, histone modification landscapes, and most compellingly, these iPS cells are capable of complementing tetraploid embryos and completely enabling the proper fetal development of tetraploid blastocysts bereft of any native pluripotent stem cells219–224 — this is the most essential operational characteristic of an authentic pluripotent stem cell. The generation of iPS cells has opened many exciting, previously inaccessible, avenues for regenerative medicine and stem cell research. For example, it is possible to reprogram keratinocytes or fibroblasts taken from a human patient’s hair or skin into iPS cells that are immunologically autologous to the patient.225,226 These “patient-specific” iPS cells can then be differentiated into clinically-relevant cell types that are also immunologically autologous to the patient from whom they were derived; thus, these iPS-derived differentiated cells could be transplanted back into the patient without fear of immune rejection — thus enabling truly autologous cell replacement therapies, as have been discussed above.4 Furthermore, patient-specific iPS cells can be used to create novel models of human diseases. For example, the etiology of familial dysautonomia in human patients lies in a mutation within the Ikbkap gene, which leads to neuronal degeneration.13 Thus, human iPS cells have been attained from familial dysautonomia patients, and these iPS cells have been differentiated into neurons which exhibit the degenerative phenotype of the disease.13 These neurons serve as an excellent in vitro model to interrogate the pathogenesis of familial dysautonomia in culture, and these diseased neurons can also serve as the basis of an in vitro screening platform to
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
60
10/12/2010
4:38 PM
Page 60
K. M. Loh et al.
screen for chemical compounds and other therapeutics that could potentially ameliorate their deteriorating phenotype.13 To this end, patientspecific human iPS cells derived from familial dysautonomia patients have indeed been used to successfully screen for chemical compounds that can rescue the degenerating iPS-derived neurons,13 thus validating the promise of this patient-specific iPS cell approach to model complex diseases and to screen for potential drug candidates. Indeed, patient-specific human iPS cell lines have been created from amyotrophic lateral sclerosis patients,16 Type 1 diabetes patients,14,15 Down syndrome patients,15 Parkinson disease patients,15 Huntington disease patients,15 and patients afflicted with other diverse diseases.15,18 The complete extent of therapeutic and translational opportunities offered by iPS cells are beyond the scope of this chapter and are more extensively reviewed elsewhere.4 For us, the most compelling question concerning iPS cells is how the process of pluripotent reprogramming occurs. How is pluripotency reconstructed at a molecular level within differentiated cells? Above, we have discussed at length that systemic architectures — ensembles of transcription factors and epigenetic regulators communicating with one another — are responsible for conferring pluripotency to a pluripotent cell. How does the synergy of these factors synthesize pluripotency within a nonpluripotent cell? Given the nascent nature of this field of research, many answers are lacking on the molecular mechanisms by which overexpression of these pluripotent transcription factors recreates pluripotency within differentiated cells. However, here we will review what is known thus far. Amongst all differentiated cells, fibroblasts are the ones that are most often reprogrammed within the laboratory (using the overexpression of Oct4, Sox2, Klf4, and c-Myc — the key reprogramming factors we have disclosed above). By definition, pluripotential reprogramming must comprised two phases: erasure of the differentiated program of the starting differentiated cell and synthesis of the pluripotent state (Fig. 5). Interestingly, it appears that the former is easier than the latter; many cells successfully censor their starting differentiating program but appear to fail to successfully instate complete pluripotency. Indeed, fibroblast markers such as Thy1 or CD13 are rapidly downregulated in 33–50% of fibroblasts several days after the induction of the reprogramming factors227,228; this is concomitant with the drastic downregulation of fibroblast genes.229 Interestingly, proliferative genes are also acutely upregulated229 — it appears that an early molecular process within
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
61
10/12/2010
4:38 PM
Page 61
Molecular Principles Underlying Pluripotency and Differentiation
Figure 5. Recreating pluripotency within differentiated cells. The process of recreating pluripotency within differentiated cells by overexpression of pluripotency transcription factors has two discrete molecular phases. The first is an initial phase of de-differentiation (bottom), which generates “partially-reprogrammed” cells which will express an incomplete complement of pluripotency genes (i.e. they do not express Nanog); many cells become stably entrapped within this partially-reprogrammed state. Rare cells may progress to complete reprogramming (probably due to upregulation of Nanog), but they can adopt either one of two fates: “fully reprogrammed” iPS cells (top left) or “mostly reprogrammed” iPS cells (top right). While both display pluripotential characteristics, only the former category is capable of tetraploid complementation. It appears that expression of genes within the Gtl2 locus is diagnostic of whether or not authentic embryonic pluripotency has been recreated.
pluripotential reprogramming entails the escape of the starting cells from the anti-proliferative controls installed within most differentiated cells, thus allowing the cells to eventually attain the immortal and infinitely-replicating condition of pluripotent cells. To this end, the reprogramming factors acutely silence the Ink4/Arf tumor suppressor locus,230,231 thus enabling rapid proliferation of the starting cells. Furthermore, fibroblasts undergoing reprogramming rapidly downregulate mesenchymal genes (i.e. Snail) and concomitantly upregulate epithelial genes (i.e. E-cadherin) — this is thought
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
62
10/12/2010
4:38 PM
Page 62
K. M. Loh et al.
to befit how mesenchymal fibroblasts could transit to an epithelial-like iPS/ES state.232,233 Thus, differentiated cells rapidly transit to a proliferative and dedifferentiated intermediate within several days. Interestingly, reprogramming often irreversibly stalls at this point, thus yielding “partially-reprogrammed” cells (Fig. 5) that are de-differentiated (they have downregulated differentiation genes) but have not yet attained authentic pluripotency; they express a subset of pluripotency genes such as Oct4 and Fgf4 but critically fail to upregulate other pluripotency genes such as Nanog and Rex1.229,234 Interestingly, the de-differentiation that has occurred up to this stage can be readily reversed; withdrawal of the reprogramming factors leads to retraction of the reprogramming process, with the cells largely reverting back to their original fibroblast state.232 Nevertheless, entrapment within the “partially reprogrammed” state is quite enduring, with few cells successfully escaping this indeterminate fate and endeavoring to reach complete authentic pluripotency.229,235 Transcriptome-wide analyses of partially-reprogrammed cells suggest that they have largely reactivated ES-expressed metabolic regulators (perhaps explaining their proliferative nature) but they have failed to extensively reactivate ES-expressed transcriptional regulators — such as the aforementioned Nanog.236 This is convergent with findings that Oct4, Sox2, and Klf4 fail to bind their common target genes within partiallyreprogrammed intermediates.236 These findings suggest that partially-reprogrammed cells become entrapped within their indeterminate state due to failure of these key pluripotency architects to bind to their target genes and re-enact the molecular program of pluripotency. Such failure perhaps could be because the chromatin state of these partially-reprogrammed cells repels the binding of these reprogramming factors to their target pluripotency genes, thus impeding reprogramming. Alternatively, it could be because in the absence of the expression of these ES-expressed transcription factors such as Nanog, the reprogramming factors (Oct4, Sox2, and Klf4) may be unable to assemble into the aforementioned “enhanceosome” complexes that are surmised to be necessary for upregulation of a subset of ES-expressed genes within ES cells. Nevertheless, rare intermediate cells progress further and faithfully reconstruct authentic embryonic pluripotency from the incomplete vestiges of the pluripotency network that are present within partiallyreprogrammed cells (Fig. 5). The molecular events that orchestrate
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
63
10/12/2010
4:38 PM
Page 63
Molecular Principles Underlying Pluripotency and Differentiation
liberation from the partially-reprogrammed state and ascension to a pluripotent state still remain unclear. Nevertheless, it appears that Nanog is key to the successful instatement of pluripotency within partially-reprogrammed cells. Specifically, Nanog-/- differentiated cells are capable of progression to the de-differentiated partially-reprogrammed state, but unequivocally fail to progress further — none can attain authentic pluripotency.179 Thus, Nanog is entirely dispensable for the initial de-differentiation phase but it is absolutely required for the final synthesis of complete pluripotency within partially-reprogrammed cells. Such a specific requirement for Nanog during reprogramming parallels its essential role in the construction of pluripotency within the pluripotent epiblast during development.44,179 To this end, direct overexpression of Nanog within partially-reprogrammed cells converts them into iPS cells.179
Recreating Authentic Embryonic Pluripotency However, of all the iPS cell lines established thus far, exceedingly few lines (only ∼5%) are likely capable of tetraploid complementation.223 The remaining ∼95% of mouse iPS cell lines have pluripotential characteristics, as they are capable of multilineage differentiation, but their impotency for tetraploid complementation disqualifies them as authentically pluripotent cells.224 From this, we conclude that overexpression of the reprogramming factors often fails to recreate authentic embryonic pluripotency in the majority of differentiated mouse cells.41 Although it appears that all mouse iPS cell lines express pluripotency markers and are capable of differentiation into cell types of all three fetal layers (as assayed by teratoma formation and in vitro differentiation), the defining characteristic of an authentic pluripotent stem cell line is that it can generate every cell type within the fetus, i.e. it can colonize a tetraploid embryo completely devoid of pluripotent stem cells, thus independently rescuing proper fetal embryogenesis and generating healthy, live-born mice. We surmise that the majority of mouse iPS cells which can activate an Oct4-GFP reporter but that are incapable of tetraploid complementation are indeed only “mostly reprogrammed,” which reflects the failure of the reprogramming factors to entirely recreate authentic pluripotency within these cells.41 The rare mouse iPS cell lines that are capable of tetraploid complementation are truly bona fide pluripotent stem cell lines, and we call them “fully
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
64
10/12/2010
4:38 PM
Page 64
K. M. Loh et al.
reprogrammed,” reflecting how in rare and infrequent instances, the reprogramming factors have succeeded in recreating an authentic, fully functional pluripotent state (Fig. 5).41 A recent study has demonstrated that extremely few genes are actually differentially expressed between these “mostly reprogrammed” iPS cell lines and tetraploid complementation-competent “fully reprogrammed” iPS cell lines. In fact, it appears that the tremendous functional disparity between these two types of iPS cells is in fact due to the differential expression of just three genes and 21 microRNAs, all of which reside within the Gtl2 locus.223 While mouse ES cell lines (all of which are capable of tetraploid complementation) and “fully reprogrammed” mouse iPS cell lines express moderate levels of transcripts from the Gtl2 locus, all “mostly reprogrammed” mouse iPS cell lines incapable of tetraploid complementation do not express transcripts from the Gtl2 locus.223 Reactivation of transcription from the Gtl2 locus in “mostly reprogrammed” mouse iPS cell lines (by treatment with a chemical inhibitor of histone deacetylases, valproic acid) makes them capable of tetraploid complementation,223 thus indicating that activation or silencing of the Gtl2 locus in mouse pluripotent stem cells is the critical determinant of whether or not they are capable of tetraploid complementation, i.e., whether or not they are authentic pluripotent stem cells.41 It is puzzling that the overexpression of the same combination of the four reprogramming factors can generate two different subsets of stable pluripotent stem cells, one of which is deficient in tetraploid complementation and the other that is authentically pluripotent. Thus, it is likely that there is some stochastic molecular process at work that defines whether or not the reprogramming factors are successful at activating the Gtl2 locus and re-instating authentic pluripotency. Interestingly, overexpression of the transcription factor Tbx3 during the reprogramming process produces mouse iPS cells which have an enhanced capacity for germline transmission and tetraploid complementation.237 Thus, Tbx3 can act in concert with the other reprogramming factors to help reconstruct an authentic pluripotency network and it enhances the efficiency with which genuine embryonic pluripotency can be attained (although it is of note that not all iPS cell lines produced with Tbx3 overexpression are capable of tetraploid complementation).237 Recently, overexpression of either c-Myc or L-Myc in conjunction with Oct4, Sox2, and Klf4 has also been shown to create mouse iPS cell lines with enhanced germline transmission.238
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
65
10/12/2010
4:38 PM
Page 65
Molecular Principles Underlying Pluripotency and Differentiation
What can we learn from these studies that informs us about the molecular reconstruction of the pluripotency network during iPS reprogramming? It appears that at least in the case of mouse iPS cells, there are two possible configurations of the pluripotency network that can be reconstructed: “mostly reprogrammed” and “fully reprogrammed.”41 Both give rise to iPS cells with superficially pluripotential characteristics — such as expression of pluripotency markers and the potential for teratoma formation — but only the latter network produces authentic pluripotent stem cells that are capable of creating every embryonic cell type (as assayed by the tetraploid complementation assay). Perhaps the only major difference between these two network configurations is that in one, genes from the Gtl2 locus are upregulated.223 Although it is currently undetermined whether or not expression of genes from the Gtl2 locus are the functional cause of the enhanced developmental potency of “fully reprogrammed” iPS cells, or if upregulation of these Gtl2 genes is simply a marker for the fully reprogrammed state, it is of particular note that Tbx3 can act as a constructor of the fully reprogrammed network, helping to recreate it. Remarkably, it appears that “higher quality” iPS cell lines produced with the overexpression of Tbx3 do not express markedly higher levels of Tbx3 versus iPS cell lines produced with the classical reprogramming factors alone.237 Thus, it appears that Tbx3 acts specifically to construct the authentic pluripotency network while it is being assembled, but active expression of Tbx3 is likely unnecessary for maintaining the “fully reprogrammed” network after it has been produced.237 Indeed, only a few genes appear to be differentially expressed between iPS cell lines produced with or without Tbx3 overexpression; Nanog is upregulated by approximately 40% and Oct4 is upregulated by approximately 20% in iPS cell lines produced with Tbx3 overexpression. Nevertheless, it should be noted that upregulation of Oct4 expression has previously been shown to be critical for the successful establishment of ES cell lines from cloned embryos.239 Taken altogether, although pluripotential reprogramming by the overexpression of transcription factors is a highly-promising approach to generate patient-specific iPS cell lines, the current combination of reprogramming factors often fails to recreate authentic embryonic pluripotency within differentiated cells. It is unknown how these reprogramming factors could produce “mostly-reprogrammed” cells with pluripotential characteristics (multilineage differentiation) but that are
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
66
10/12/2010
4:38 PM
Page 66
K. M. Loh et al.
ultimately incapable of tetraploid complementation. Nevertheless, overexpression of at least one transcription factor — Tbx3 — has been shown to assist the recreation of authentic pluripotency (although its molecular mechanism of action remains to be determined). We remain confident that in the near future, additional factors will be identified that help recreate genuine pluripotency, thus allowing for the efficient production of authentically pluripotent iPS cell lines from differentiated cells.
Novel Architects of Pluripotency Another focus of reprogramming research has been finding alternative combinations of transcription factors that can reconstruct the pluripotent state within differentiated cells. Such an approach has been innovatively called a “gain of function” test to identify factors that sustain pluripotency240; logically, if a factor can reconstruct the pluripotency transcriptional network in a non-pluripotent cell, it is likely that it also safeguards the pluripotency network within existing pluripotent cells such as ES cells (although it should be noted that this is not always the case).179,241 Although the original combination of transcription factors that was found to reprogram fibroblasts into iPS cells consisted of Oct4, Sox2, Klf4, and c-Myc,47 it was found shortly thereafter that c-Myc was dispensable for pluripotent reprogramming, and thus, Oct4, Sox2, and Klf4 have been highlighted as the classic trio of reprogramming factors that can reprogram both human and mouse fibroblasts and many other cell types into iPS cells.242,243 Oct4, Sox2, and Nanog or Oct4, Sox2, and Lin28 are also capable of reprogramming human fibroblasts into iPS cells,49 thus indicating that overexpression of either Lin28 and Nanog can functionally “replace” the requisite for Klf4 overexpression during reprogramming. Additional experiments have found that overexpression of the orphan nuclear receptors Nr5a2 or Nr5a1 can replace Oct4,244 that overexpression of transcription factors Nanog, Sox1, Sox3, or Sox15 can replace Sox2,242,245 and that overexpression of the orphan nuclear receptors Esrrb or Esrrg 246 or overexpression of transcription factors Klf2 or Klf5 can also replace Klf4 242 (summarized in Table 2). While the Oct4, Sox2, and Klf4 combination is often the minimal requirement to reprogram most differentiated cell types, overexpression of all three factors is not necessary to reprogram cell types to already express some of
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
67
10/12/2010
4:38 PM
Page 67
Molecular Principles Underlying Pluripotency and Differentiation Table 2. Overview of Minimal Combinations of Factors Required for Pluripotent Reprogramming
Transcription Factors OCT4, SOX2, and KLF4† NR5A2, SOX2, and KLF4 NR5A1, SOX2, and KLF4 OCT4, NANOG, and KLF4 OCT4, SOX1, and KLF4 OCT4, SOX3, and KLF4 OCT4, SOX15, and KLF4 OCT4, TGFβ inhibitors, and KLF4 OCT4, GSK3 inhibitor, and KLF4 OCT4, G9a HMTase inhibitor, and KLF4 OCT4, SOX2, and NANOG† OCT4, SOX2, and LIN28† OCT4, SOX2, and ESRRB OCT4, SOX2, and ESRRG OCT4, SOX2, and KLF2 OCT4, SOX2, and KLF5 OCT4, SOX2, and kenpaullone OCT4, SOX2, and HDAC inhibitor†
References Nakagawa et al. (2008)242 Heng et al. (2010)244 Heng et al. (2010)244 Ichida et al. (2009)245 Nakagawa et al. (2008)242 Nakagawa et al. (2008)242 Nakagawa et al. (2008) Ichida et al. (2009)245; Maherali et al. (2009)258 Li et al. (2009)260 Shi et al. (2008)259 Yu et al. (2007)49 Yu et al. (2007)49 Feng et al. (2009)246 Feng et al. (2009)246 Nakagawa et al. (2008)242 Nakagawa et al. (2008)242 Lyssiotis et al. (2009)261 Huangfu et al. (2008)262
Red, blue, and green indicate transcription factors or chemical compounds that can “replace” the requirement for OCT4, SOX2, and KLF4 (respectively) in the pluripotent reprogramming of mouse fibroblasts. Dagger indicates that the combination of factors can reprogram human fibroblasts. TGFβ; transforming growth factor-β. GSK3; glycogen synthase kinase 3. HMTase; histone methyltransferase. HDAC; histone deacetylase.
these genes. For example, the pluripotent reprogramming of neural stem cells only requires the overexpression of Oct4, as these stem cells already express Sox2 and Klf4.247 Conversely, some terminally-differentiated cell types appear to be more intractable to lineage reprogramming, and they require additional extrinsic coercion besides overexpression of these classical reprogramming factors in order to elicit their pluripotent reprogramming. For example, the efficient pluripotent reprogramming of terminally-differentiated B lymphocytes with these reprogramming factors requires the destabilization of the firmly-fixated B cell lineage program by concomitant knockdown of the B cell transcription factor Pax5 or the co-overexpression of the transcription factor C/EBPα.218,248 Interestingly, chemical compounds have also been found to replace the need to overexpress transcription factors by viral vectors in order to
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
68
10/12/2010
4:38 PM
Page 68
K. M. Loh et al.
reprogram cells viral transduction is a hazardous procedure that leads to the integration of proviruses into the genomes of the resultant iPS cells. Proviral integrations often activate proximal oncogenes within the genome, thus leading to oncogenic transformation of the transduced cells.249,250 To this end, it is held that iPS cells produced using integrating vectors are unfit for therapeutic usage in humans.4,251 In order to produce iPS cells for eventual therapeutic usage, it will be necessary to find alternative methods to deliver the reprogramming factors to the target cells besides the integrating vectors. Several methods have been proposed, including non-integrating adenoviruses,252 self-excising lentiviruses,253 excisable piggyBac transposons,254 and transient plasmid transfection.255 Nevertheless, all these approaches still have drawbacks, including a latent risk of producing some genomic integrations.4,256 Using chemical compounds to reprogram cells instead of genetic vectors would completely ensure that no insertional mutagenesis was elicited during the reprogramming process, thus producing genetically modified iPS cell lines competent for usage in human patients. Additionally, identi-fication of the cellular proteins that are either activated or inhibited by these reprogramming chemicals would immediately implicate a role for those proteins in pluripotent reprogramming, thus allowing for elucidation of the molecular mechanisms that underlie reprogramming.245,257 To this end, overexpression of Sox2 in mouse fibroblasts can be bypassed by treatment with chemical inhibitors of TGF-β signaling,245,258 treatment with a chemical inhibitor of the G9a histone methyltransferase,259 or treatment with a chemical inhibitor of GSK3.260 Overexpression of Klf4 in mouse fibroblasts can be replaced by treatment with a nonspecific kinase inhibitor (kenpaullone),261 and the overexpression of Klf4 in human fibroblasts can be bypassed by treatment with a chemical inhibitor of histone deacetylases (valproic acid).262 These minimal combinations of transcription factors and chemical compounds that can elicit pluripotent reprogramming are summarized in Table 2. We conclude by reiterating that pluripotential reprogramming is not only of academic interest — a cellular sleight of hand previously thought impossible. Pluripotent reprogramming offers avenues to ethically create pluripotent stem cells bereft of the embryo. The capability to generate iPS cells from adult tissues negotiates the traditional ethical issue with pluripotent stem cells that human ES cell lines must be derived from
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
69
10/12/2010
4:38 PM
Page 69
Molecular Principles Underlying Pluripotency and Differentiation
embryonic tissues (although it should be noted that iPS cells themselves have unique moral, ethical, and legal problems inherent to them).43,263 Furthermore, the production of patient-specific iPS cell lines from human patients will empower autologous cell replacement therapies for these patients and it will also enable the construction of novel in vitro models of human diseases.
Conclusion — ¡Viva la Pluripotency! Here, we have discussed the molecular principles that underlie the pluripotency and differentiation of pluripotent stem cells. Exploitation of the pluripotent and the indefinitely-replicating nature of embryonic stem cells and induced pluripotent stem cells will allow us to produce prodigious numbers of clinically-relevant cell types (such as pancreatic β-cells) for cell replacement therapies. Furthermore, the opportunity afforded by pluripotent stem cells for us to observe and model embryonic development in cell culture has also enabled us to make several unanticipated insights about embryogenesis that would otherwise be difficult to perceive if we had utilized traditional in vivo approaches.10,12 Pluripotent stem cells have already enabled us to make key advances in regenerative medicine and developmental biology, and we predict that continued exploitation of such stem cells will lead to further breakthroughs in the future. Compellingly, studies of the embryonic epiblast and its explanted derivatives (embryonic stem cells) have also yielded novel insights into the molecular nature of pluripotency — a remarkable physiological ability that is indispensible for embryonic development, yet something we still know so little about. Here, we have briefly reviewed known transcription factors and epigenetic regulators that safeguard pluripotency and we have also advanced several novel theses concerning the molecular nature of pluripotency. Despite assertions that the pluripotency of embryonic stem cells is stable,264 here we have highlighted that key pluripotency transcription factors upregulate differentiation genes (such as Fgf4 and Tcf3) while driving their own expression,163–168 suggesting that pluripotency is transcriptionally constructed to be poised for collapse into differentiation. We also have presented a working model to explain the unrestricted lineage potency of embryonic stem cells by discussing how certain
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
70
10/12/2010
4:38 PM
Page 70
K. M. Loh et al.
pluripotency transcription factors (such as Oct4, Sox2, and Esrrb) drive differentiation into particular lineages and that slight upregulation of such factors could drive stem cell differentiation into those specific lineages. Finally, we have defended the important role of functional communication between transcription factors and epigenetic regulators in safeguarding pluripotency. Identification of the molecular ensembles that underlie pluripotency has also allowed for the artificial recreation of pluripotency within differentiated cells by overexpression of pluripotency transcription factors — many of the same factors that safeguard pluripotency can also recreate it within differentiated cells. The production of induced pluripotent stem cells has negotiated certain ethical and moral issues pertaining to the derivation of embryonic stem cells and it also offers new opportunities for patient-specific cell replacement therapies and disease modeling. The finding that pluripotential reprogramming is possible has also galvanized the field of direct reprogramming — that is, the lineage reprogramming of differentiated cells directly into another differentiated cell type (instead of back to a pluripotent state). Recent decisive advances in the field of direct reprogramming include the in vivo reprogramming of cochlear cells into auditory inner hair cells, thus reversing deafness within mature animals,265 and the in vivo reprogramming of pancreatic α-cells into insulin-secreting β-cells, thus ameliorating hyperglycemia within diabetic mice models.266 Such amazing demonstrations of the therapeutic utility of direct reprogramming suggest that cell replacement therapy within human patients can be done one day by simply reprogramming cells within the patient’s body, thus circumventing the need to transplant cells into a patient’s body as would be required if a pluripotent stem cellbased therapy was used.267 We conclude that capitalization on the potential of pluripotent stem cells will further our understanding of developmental biology and will enable new strategies for regenerative medicine.
Acknowledgments We thank Siming Ma for his critical review of the manuscript and for provocative discussions. We also thank Guoji Guo, Yixuan Wu, Swea Ling Khaw, Li Pin, Shaun Lim, and other members of our laboratory for insightful discussions. K.M.L. is supported by a fellowship from the
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
71
10/12/2010
4:38 PM
Page 71
Molecular Principles Underlying Pluripotency and Differentiation
Davidson Institute for Talent Development and by a Singapore International Pre-Graduate Award from the Singapore Agency for Science, Technology and Research (A*STAR). B.S.S. and W.L.T. are recipients of graduate scholarships from the Singapore Agency for Science, Technology, and Research (A*STAR). This work is supported by A*STAR and it is also partially supported by U.S. National Institutes of Health grants to B.L. (DK047636 and AI54973). We also apologize to all of our colleagues in the field whose work we were unable to cite due to space constraints. References 1. Thomas L. (1995) The Medusa and the Snail: More Notes of a Biology Watcher, 175 (Penguin Books). 2. Nichols J, Smith A. (2009) Naive and primed pluripotent states. Cell Stem Cell 4: 487–492. 3. Gardner RL, Rossant J. (1979) Investigation of the fate of 4.5 day postcoitum mouse inner cell mass cells by blastocyst injection. J Embryol Exp Morphol 52: 141–152. 4. Yamanaka S. (2009) A fresh look at iPS cells. Cell 137: 13–17. 5. Murry CE, Keller G. (2008) Differentiation of embryonic stem cells to clinically relevant populations: Lessons from embryonic development. Cell 132: 661–680. 6. Pera MF, Trounson AO. (2004) Human embryonic stem cells: Prospects for development. Development 131: 5515–5525. 7. Kiskinis E, Eggan K. (2010) Progress toward the clinical application of patient-specific pluripotent stem cells. J Clin Invest 120: 51–59. 8. Keller G. (2005) Embryonic stem cell differentiation: Emergence of a new era in biology and medicine. Genes Develop 19: 1129–1155. 9. Nishikawa S-I, Jakt LM, Era T. (2007) Embryonic stem-cell culture as a tool for developmental cell biology. Nat Revi Mol Cell Biol 8: 502–507. 10. Chen S, et al. (2009) A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat Chem Biol 5: 258–265. 11. Bu L, et al. (2009) Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature 460: 113–117. 12. Kennedy M, et al. (1997) A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature 386: 488–493. 13. Lee G, et al. (2009) Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461: 402–406. 14. Maehr R, et al. (2009) Generation of pluripotent stem cells from patients with type 1 diabetes. Proc Natl Acad Sci USA 106: 15768–15773.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
72
10/12/2010
4:38 PM
Page 72
K. M. Loh et al.
15. Park I-H, et al. (2008) Disease-specific induced pluripotent stem cells. Cell 134: 877–886. 16. Dimos JT, et al. (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321: 1218–1221. 17. Carvajal-Vergara X, et al. (2010) Patient-specific induced pluripotent stemcell-derived models of LEOPARD syndrome. Nature 465: 808–812. 18. Ebert AD, et al. (2009) Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457: 277–280. 19. Kroon E, et al. (2008) Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol 26: 443–452. 20. Laflamme MA, et al. (2007) Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol 25: 1015–1024. 21. Beddington RS, Morgernstern J, Land H, Hogan A. (1989) An in situ transgenic enzyme marker for the midgestation mouse embryo and the visualization of inner cell mass clones during early organogenesis. Development 106: 37–46. 22. Švajger A, Levak-Švajger B, Škreb N. (1986) Rat embryonic ectoderm as renal isograft. J Embryol Exp Morphol 94: 1–27. 23. Arnold SJ, Robertson EJ. (2009) Making a commitment: Cell lineage allocation and axis patterning in the early mouse embryo. Nat Revi Mol Cell Biol 10: 91–103. 24. Tam PPK, Loebel DAF. (2007) Gene function in mouse embryogenesis: Get set for gastrulation. Nat Revi Genet 8: 368–381. 25. Lawson KA, Meneses JJ, Pedersen RA. (1991) Clonal analysis of epiblast fate during germ layer formation in the mouse embryo. Development 113: 891–911. 26. Evans MJ, Kaufman MH. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292: 154–156. 27. Martin GR. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78: 7634–7638. 28. Brook FA, Gardner RL. (1997) The origin and efficient derivation of embryonic stem cells in the mouse. Proc Natl Acad Sci USA 94: 5709–5712. 29. Tang F, et al. (2010) Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis. Cell Stem Cell 6: 468–478. 30. Nagy A, et al. (1990) Embryonic stem cells alone are able to support fetal development in the mouse. Development 110: 815–821. 31. Nagy A, Rossant J, Nagy R, et al. (1993) Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci USA 90: 8424–8428.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
73
10/12/2010
4:38 PM
Page 73
Molecular Principles Underlying Pluripotency and Differentiation
32. Brons IGM, et al. (2007) Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448: 191–195. 33. Tesar PJ, et al. (2007) New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448: 196–199. 34. Tam PPL, Loebel DAF, Tanaka SS. (2006) Building the mouse gastrula: Signals, asymmetry and lineages. Curr Opin Genet Dev 16: 419–425. 35. Guo G, et al. (2009) Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136: 1063–1069. 36. Bao S, et al. (2009) Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells. Nature 461: 1292–1295. 37. Pera MF, Tam PPL. (2010) Extrinsic regulation of pluripotent stem cells. Nature 465: 713–720. 38. Klimanskaya I, Chung Y, Becker S, et al. (2006) Human embryonic stem cell lines derived from single blastomeres. Nature 444: 481–485. 39. Thomson JA, et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282: 1145–1147. 40. Reubinoff BE, Pera MF, Fong CY, et al. (2000) Embryonic stem cell lines from human blastocysts: Somatic differentiation in vitro. Nat Biotechnol 18: 399–404. 41. Loh KM, Lim B. (2010) Recreating pluripotency? Cell Stem Cell 7: 137–139. 42. Chung Y, et al. (2006) Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres. Nature 439: 216–219. 43. Hyun I. (2010) The bioethics of stem cell research and therapy. J Clin Invest 120: 71–75. 44. Mitsui K, et al. (2003) The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113: 631–642. 45. Hochedlinger K, Jaenisch R. (2002) Monoclonal mice generated by nuclear transfer from mature B, T donor cells. Nature 415: 1035–1038. 46. Eggan K, et al. (2004) Mice cloned from olfactory sensory neurons. Nature 428, 44–49. 47. Takahashi K, Yamanaka S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676. 48. Takahashi K, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861–872. 49. Yu J, et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917–1920. 50. Shamblott MJ, et al. (1998) Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci USA 95: 13726–13731. 51. Matsui Y, Zsebo K, Hogan BL (1992). Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70: 841–847.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
74
10/12/2010
4:38 PM
Page 74
K. M. Loh et al.
52. Finch BW, Ephrussi B. (1967) Retention of multiple potentialities by cells of a mouse testicular teratocarcinoma during prolonged culture in vitro and their extinction upon hybridization with cells of permanent lines. Proc Natl Acad Sci USA 57: 615–621. 53. Brinster RL. (1974) The effect of cells transferred into the mouse blastocyst on subsequent development. J Exp Med 140: 1049–1056. 54. Kanatsu-Shinohara M, et al. (2004) Generation of pluripotent stem cells from neonatal mouse testis. Cell 119: 1001–1012. 55. Lerou PH, Daley GQ. (2005) Therapeutic potential of embryonic stem cells. Blood Rev 19: 321–331. 56. Smith A. (1998) Cell therapy: In search of pluripotency. Curr Biol 8: R802–R804. 57. Lamba DA, Gust J, Reh TA. (2009) Transplantation of human embryonic stem cell-derived photoreceptors restores some visual function in Crxdeficient mice. Cell Stem Cell 4: 73–79. 58. Rideout WM, Hochedlinger K, Kyba M, et al. (2002) Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 109: 17–27. 59. Wernig M, et al. (2008) Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease. Proc Natl Acad Sci USA 105: 5856–5861. 60. Roy NS, et al. (2006) Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat Med 12: 1259–1268. 61. Keirstead HS, et al. (2005) Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci 25: 4694–4705. 62. Irion S, et al. (2007) Identification and targeting of the ROSA26 locus in human embryonic stem cells. Nat Biotechnol 25: 1477–1482. 63. Thomas KR, Capecchi MR. (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51: 503–512. 64. Schaniel C, et al. (2006) Delivery of short hairpin RNAs — triggers of gene silencing — into mouse embryonic stem cells. Nat Methods 3: 397–400. 65. Chambers I, et al. (2003) Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113: 643–655. 66. Pritsker M, Ford NR, Jenq HT, Lemischka IR. (2006) Genomewide gain-offunction genetic screen identifies functionally active genes in mouse embryonic stem cells. Proc Natl Acad Sci USA 103: 6946–6951. 67. Abujarour R, Efe J, Ding S. (2010) Genome-wide gain-of-function screen identifies novel regulators of pluripotency. Stem Cells (advance online publications).
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
75
10/12/2010
4:38 PM
Page 75
Molecular Principles Underlying Pluripotency and Differentiation
68. Aubert J, Dunstan H, Chambers I, Smith A. (2002) Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. Nat Biotechnol 20: 1240–1245. 69. Chen S, et al. (2006) Self-renewal of embryonic stem cells by a small molecule. Proc Natl Acad Sci USA 103: 17266–17271. 70. Ding L, et al. (2009) A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity. Cell Stem Cell 4: 403–415. 71. Xu Y, Shi Y, Ding S. (2008) A chemical approach to stem-cell biology and regenerative medicine. Nature 453: 338–344. 72. Miller CL, Eaves CJ. (1997) Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability. Proc Natl Acad Sci USA 94: 13648–13653. 73. Hope KJ, et al. (2010) An RNAi screen identifies Msi2 and Prox1 as having opposite roles in the regulation of hematopoietic stem cell activity. Cell Stem Cell 7: 101–113. 74. Tam PPL, Loebel DAF. (2007) Gene function in mouse embryogenesis: Get set for gastrulation. Nat Rev Genet 8: 368–381. 75. Wells JM, Melton DA. (1999) Vertebrate endoderm development. Annu Rev Cell Dev Biol 15: 393–410. 76. Grapin-Botton A, Melton DA. (2000) Endoderm development: From patterning to organogenesis. Trends in Genet 16: 124–130. 77. Kim SK, MacDonald RJ. (2002) Signaling and transcriptional control of pancreatic organogenesis. Curr Opin Genet Dev 12: 540–547. 78. Wichterle H, Lieberam I, Porter JA, Jessell TM. (2002) Directed differentiation of embryonic stem cells into motor neurons. Cell 110: 385–397. 79. D’amour KA, et al. (2006) Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 24: 1392–1401. 80. Loebel DAF, Watson CM, De Young RA, Tam PPL. (2003) Lineage choice and differentiation in mouse embryos and embryonic stem cells. Dev Biol 264: 1–14. 81. Gouon-Evans V, et al. (2006) BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nat Biotechnol 24: 1402–1411. 82. Yang L, et al. (2008) Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453: 524–528. 83. Levenberg S, Golub JS, Amit M, et al. (2002). Endothelial cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 99: 4391–4396. 84. Elkabetz Y, et al. (2008) Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Develop 22: 152–165.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
76
10/12/2010
4:38 PM
Page 76
K. M. Loh et al.
85. Perrier AL, et al. (2004) Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci USA 101: 12543–12548. 86. Yan Y, et al. (2005) Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 23: 781–790. 87. Di Giorgio FP, Boulting GL, Bobrowicz S, Eggan KC. (2008) Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation. Cell Stem Cell 3: 637–648. 88. Nistor GI, Totoiu MO, Haque N, et al. (2005) Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation. Glia 49: 385–396. 89. Meyer JS, et al. (2009) Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc Natl Acad Sci USA 106: 16698–16703. 90. Ito K, Sieber-Blum M. (1993) Pluripotent and developmentally restricted neural-crest-derived cells in posterior visceral arches. Dev Biol 156: 191–200. 91. Baroffio A, Dupin E, Le Douarin NM. (1988) Clone-forming ability and differentiation potential of migratory neural crest cells. Proc Natl Acad Sci USA 85: 5325–5329. 92. Takashima Y, et al. (2007) Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 129: 1377–1388. 93. D’amour KA, et al. (2005) Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 23: 1534–1541. 94. Beddington RSP. (1981) An autoradiographic analysis of the potency of embryonic ectoderm in the 8th day postimplantation mouse embryo. J Embryol Exp Morphol 64: 87–104. 95. Tada S, et al. (2008) Characterization of mesendoderm: A diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 132: 4363–4374. 96. Kimelman D, Griffin KJ. (2000) Vertebrate mesendoderm induction and patterning. Curr Opin Genet Dev 10: 350–356. 97. Kubo A, et al. (2004) Development of definitive endoderm from embryonic stem cells in culture. Development 131: 1651–1662. 98. Gadue P, Huber TL, Paddison PJ, Keller GM. (2006) Wnt and TGF-β signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells. Proc Natl Acad Sci USA 103: 16806–16811. 99. Nostro MC, Cheng X, Keller GM, Gadue P. (2008) Wnt, activin, and BMP signaling regulate distinct stages in the developmental pathway from embryonic stem cells to blood. Cell Stem Cell 2: 60–71.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
77
10/12/2010
4:38 PM
Page 77
Molecular Principles Underlying Pluripotency and Differentiation
100. Ueno S, et al. (2007) Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells. Proc Natl Acad Sci USA 104: 9685–9690. 101. Borowiak M, et al. (2009) Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells. Cell Stem Cell 4: 348–358. 102. Graichen R, et al. (2008) Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK. Differentiation 76: 357–370. 103. Smith JR, et al. (2008) Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm. Dev Biol 313: 107–117. 104. Kawasaki H, et al. (2000) Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28: 31–40. 105. Ying Q-L, Stavridis M, Griffiths D, Li M, et al. (2003) Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 21: 183–186. 106. Chambers SM, et al. (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27: 275–280. 107. Lawson KA, Meneses JJ, Pedersen RA. (1986) Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer. Dev Biol 115: 325–339. 108. Jung J, Zheng M, Goldfarb M, Zaret KS. (1999) Initiation of mammalian liver development from endoderm by fibroblast growth factors. Science 284: 1998–2003. 109. Rossi JM, Dunn NR, Hogan BL, Zaret KS. (2001) Distinct mesodermal signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm. Genes Develop 15: 1998–2009. 110. Stafford D, et al. (2006) Retinoids signal directly to zebrafish endoderm to specify insulin-expressing beta-cells. Development 133: 949–956. 111. Stafford D, Hornbruch A, Mueller PR, Prince VE. (2004) A conserved role for retinoid signaling in vertebrate pancreas development. Dev Genes Evol 214: 432–441. 112. Stafford D, Prince VE. (2002) Retinoic acid signaling is required for a critical early step in zebrafish pancreatic development. Curr Biol 12: 1215–1220. 113. Kim SK, Melton DA. (1998) Pancreas development is promoted by cyclopamine, a hedgehog signaling inhibitor. Proc Natl Acad Sci USA 95: 13036–13041. 114. Bhushan A, et al. (2001) Fgf10 is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis. Development 128: 5109–5117.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
78
10/12/2010
4:38 PM
Page 78
K. M. Loh et al.
115. Lickert H, et al. (2002) Formation of multiple hearts in mice following deletion of beta-catenin in the embryonic endoderm. Dev Cell 3: 171–181. 116. Tzahor E, Lassar AB. (2001) Wnt signals from the neural tube block ectopic cardiogenesis. Genes Develop 15: 255–260. 117. Marvin MJ, Di Rocco G, Gardiner A, et al. (2001) Inhibition of Wnt activity induces heart formation from posterior mesoderm. Genes Develop 15: 316–327. 118. Naito AT, et al. (2006) Developmental stage-specific biphasic roles of Wnt/beta-catenin signaling in cardiomyogenesis and hematopoiesis. Proc Natl Acad Sci USA 103: 19812–19817. 119. Kehat I, et al. (2004) Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol 22: 1282–1289. 120. Drukker M, Benvenisty N. (2004) The immunogenicity of human embryonic stem-derived cells. Trends Biotechnol 22: 136–141. 121. Daley GQ, Scadden DT. (2008) Prospects for stem cell-based therapy. Cell 132, 544–548. 122. Daley GQ, et al. (2009) Broader implications of defining standards for the pluripotency of iPSCs. Cell Stem Cell 4: 200–201. 123. Chien KR, Domian IJ, Parker KK. (2008) Cardiogenesis and the complex biology of regenerative cardiovascular medicine. Science 322: 1494–1497. 124. Feinberg AW, et al. (2007) Muscular thin films for building actuators and powering devices. Science 317: 1366–1370. 125. Radisic M, et al. (2004) Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proc Natl Acad Sci USA 101: 18129–18134. 126. Madden LR, et al. (2010) Proangiogenic scaffolds as functional templates for cardiac tissue engineering. Proc Natl Acad Sci USA 107: 15211–15216. 127. Egli D, Birkhoff G, Eggan K. (2008) Mediators of reprogramming: Transcription factors and transitions through mitosis. Nat Rev Mol Cell Biol 9: 505–516. 128. Johnson KM, Mitsouras K, Carey M. (2001) Eukaryotic transcription: The core of eukaryotic gene activation. Curr Biol 11: R510–513. 129. Agalioti T, et al. (2000) Ordered recruitment of chromatin modifying and general transcription factors to the IFN-beta promoter. Cell 103: 667–678. 130. Métivier R, et al. (2003) Estrogen receptor-α directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115: 751–763. 131. Chambers I, Tomlinson SR. (2009) The transcriptional foundation of pluripotency. Development 136: 2311–2322. 132. Silva J, Smith A. (2008) Capturing pluripotency. Cell 132: 532–536.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
79
10/12/2010
4:38 PM
Page 79
Molecular Principles Underlying Pluripotency and Differentiation
133. Jonsson J, Carlsson L, Edlund T, Edlund H. (1994) Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371: 606–609. 134. Koopman P, Gubbay J, Vivian N, et al. (1991) Male development of chromosomally female mice transgenic for Sry. Nature 351: 117–121. 135. Uhlenhaut NH, et al. (2009) Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. Cell 139: 1130–1142. 136. Nichols J, et al. (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95: 379–391. 137. Avilion AA, et al. (2003) Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Develop 17: 126–140. 138. Schöler HR, Dressler GR, Balling R, et al. (1990) Oct-4: A germline-specific transcription factor mapping to the mouse t-complex. EMBO J 9: 2185–2195. 139. Rosner MH, et al. (1990) A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345: 686–692. 140. Initiative ISC, et al. (2007) Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol 25: 803–816. 141. Ivanova N, et al. (2006) Dissecting self-renewal in stem cells with RNA interference. Nature 442: 533–538. 142. Zhang X, Zhang J, Wang T, et al. (2008) Esrrb activates Oct4 transcription and sustains self-renewal and pluripotency in embryonic stem cells. J Biol Chem 283: 35825–35833. 143. Ema M, et al. (2008) Krüppel-like factor 5 is essential for blastocyst development and the normal self-renewal of mouse ESCs. Cell Stem Cell 3: 555–567. 144. Hanna LA, Foreman RK, Tarasenko IA, et al. (2002) Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo. Genes Develop 16: 2650–2661. 145. Cartwright P, et al. (2005) LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development 132: 885–896. 146. Galan-Caridad JM, et al. (2007) Zfx controls the self-renewal of embryonic and hematopoietic stem cells. Cell 129: 345–357. 147. Dejosez M, et al. (2008) Ronin is essential for embryogenesis and the pluripotency of mouse embryonic stem cells. Cell 133: 1162–1174. 148. Zhang J, et al. (2006) Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1. Nat Cell Biol 8: 1114–1123. 149. Niwa H, Miyazaki J, Smith AG. (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24: 372–376. 150. Masui S, et al. (2007) Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol 9: 625–635.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
80
10/12/2010
4:38 PM
Page 80
K. M. Loh et al.
151. Kopp JL, Ormsbee BD, Desler M, Rizzino A. (2008) Small increases in the level of Sox2 trigger the differentiation of mouse embryonic stem cells. Stem Cells 26: 903–911. 152. Teng L, Mundell NA, Frist AY, et al. (2008) Requirement for Foxd3 in the maintenance of neural crest progenitors. Development 135: 1615–1624. 153. Segre JA, Bauer C, Fuchs E. (1999) Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat Genet 22: 356–360. 154. Lensch MW, Ince TA. (2007) The terminology of teratocarcinomas and teratomas. Nat Biotechnol 25: 1211. 155. Lensch MW, Schlaeger TM, Zon LI, Daley GQ. (2007) Teratoma formation assays with human embryonic stem cells: A rationale for one type of human-animal chimera. Cell Stem Cell 1: 253–258. 156. Okumura-Nakanishi S, Saito M, Niwa H, Ishikawa F. (2005) Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells. J Biol Chem 280: 5307–5317. 157. Rodda DJ, et al. (2005) Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem 280: 24731–24737. 158. Kuroda T, et al. (2005) Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol Cell Biol 25: 2475–2485. 159. Chew J-L, et al. (2005) Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol Cell Biol 25: 6031–6046. 160. Boyer LA, et al. (2005) Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122: 947–956. 161. Yuan H, Corbi N, Basilico C, Dailey L. (1995) Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3. Genes Develop 9: 2635–2645. 162. Ambrosetti DC, Schöler HR, Dailey L, Basilico C. (2000) Modulation of the activity of multiple transcriptional activation domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer. J Biol Chem 275: 23387–2397. 163. Ambrosetti DC, Basilico C, Dailey L. (1997) Synergistic activation of the fibroblast growth factor 4 enhancer by Sox2 and Oct-3 depends on proteinprotein interactions facilitated by a specific spatial arrangement of factor binding sites. Mol Cell Biol 17: 6321–6329. 164. Kunath T, et al. (2007) FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development 134: 2895–2902. 165. Matoba R, et al. (2006) Dissecting Oct3/4-regulated gene networks in embryonic stem cells by expression profiling. PLoS ONE 1: e26. 166. Pereira L, Yi F, Merrill BJ. (2006) Repression of Nanog gene transcription by Tcf3 limits embryonic stem cell self-renewal. Mol Cell Biol 26: 7479–7491.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
81
10/12/2010
4:38 PM
Page 81
Molecular Principles Underlying Pluripotency and Differentiation
167. Tam W-L, et al. (2008) T-cell factor 3 regulates embryonic stem cell pluripotency and self-renewal by the transcriptional control of multiple lineage pathways. Stem Cells 26: 2019–2031. 168. Cole MF, Johnstone SE, Newman JJ, et al. (2008) Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells. Genes Develop 22: 746–755. 169. Ivanova NB, et al. (2002) A stem cell molecular signature. Science 298: 601–604. 170. Ramalho-Santos M, Yoon S, Matsuzaki Y, et al. (2002) “Stemness”: Transcriptional profiling of embryonic and adult stem cells. Science 298: 597–600. 171. Brandenberger R, et al. (2004) Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation. Nat Biotechnol 22: 707–716. 172. Loh Y-H, et al. (2006) The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38: 431–440. 173. Zhou Q, Chipperfield H, Melton DA, Wong WH. (2007) A gene regulatory network in mouse embryonic stem cells. Proc Natl Acad Sci USA 104: 16438–16443. 174. Chen X, et al. (2008) Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133: 1106–1117. 175. Kim J, Chu J, Shen X, et al. (2008) An extended transcriptional network for pluripotency of embryonic stem cells. Cell 132: 1049–1061. 176. Niwa H, Ogawa K, Shimosato D, Adachi K. (2009) A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature 460: 118–122. 177. Chambers I, et al. (2007) Nanog safeguards pluripotency and mediates germline development. Nature 450: 1230–1234. 178. Solter D, Knowles BB. (1978) Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci USA 75: 5565–5569. 179. Silva J, et al. (2009) Nanog is the gateway to the pluripotent ground state. Cell 138: 722–737. 180. Toyooka Y, Shimosato D, Murakami K, et al. (2008) Identification and characterization of subpopulations in undifferentiated ES cell culture. Development 135: 909–918. 181. Hayashi K, Lopes SM, Tang F, Surani MA. (2008) Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem Cell 3: 391–401. 182. Haub O, Goldfarb M. (1991) Expression of the fibroblast growth factor-5 gene in the mouse embryo. Development 112: 397–406. 183. Smith A. (2009) Design principles of pluripotency. EMBO Mol Med 1: 251–254.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
82
10/12/2010
4:38 PM
Page 82
K. M. Loh et al.
184. Shi W, et al. (2006) Regulation of the pluripotency marker Rex-1 by Nanog and Sox2. J Biol Chem 281: 23319–23325. 185. Ying QL, Nichols J, Chambers I, Smith A. (2003) BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell selfrenewal in collaboration with STAT3. Cell 115: 281–292. 186. Darr H, Mayshar Y, Benvenisty N. (2006) Overexpression of NANOG in human ES cells enables feeder-free growth while inducing primitive ectoderm features. Development 133: 1193–1201. 187. Sharova LV, et al. (2007) Global gene expression profiling reveals similarities and differences among mouse pluripotent stem cells of different origins and strains. Dev Biol 307: 446–459. 188. Eckfeldt CE, Mendenhall EM, Verfaillie CM. (2005) The molecular repertoire of the ‘almighty’ stem cell. Nat Rev Mol Cell Biol 6: 726–737. 189. Efroni S, et al. (2008) Global transcription in pluripotent embryonic stem cells. Cell Stem Cell 2: 437–447. 190. Meshorer E, et al. (2006) Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells. Dev Cell 10: 105–116. 191. Kimura H, Tada M, Nakatsuji N, Tada T. (2004) Histone code modifications on pluripotential nuclei of reprogrammed somatic cells. Mol Cell Biol 24: 5710–5720. 192. Wen B, Wu H, Shinkai Y, et al. (2009) Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells. Nat Genet 41: 246–250. 193. Ho L, Crabtree GR. (2010) Chromatin remodelling during development. Nature 463: 474–484. 194. Côté J, Quinn J, Workman JL, Peterson CL. (1994) Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science 265: 53–60. 195. Bultman S, et al. (2000) A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes. Mol Cell 6: 1287–1295. 196. Kim JK, et al. (2001) Srg3, a mouse homolog of yeast SWI3, is essential for early embryogenesis and involved in brain development. Mol Cell Biol 21: 7787–7795. 197. Stopka T, Skoultchi AI. (2003) The ISWI ATPase Snf2h is required for early mouse development. Proc Natl Acad Sci USA 100: 14097–14102. 198. Klochendler-Yeivin A, et al. (2000) The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression. EMBO Rep 1: 500–506. 199. Gaspar-Maia A, et al. (2009) Chd1 regulates open chromatin and pluripotency of embryonic stem cells. Nature 460: 863–868.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
83
10/12/2010
4:38 PM
Page 83
Molecular Principles Underlying Pluripotency and Differentiation
200. Gao X, et al. (2008) ES cell pluripotency and germ-layer formation require the SWI/SNF chromatin remodeling component BAF250a. Proc Natl Acad Sci USA 105: 6656–6661. 201. Tsumura A, et al. (2006) Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells 11: 805–814. 202. Jackson M, et al. (2004) Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells. Mol Cell Biol 24: 8862–8871. 203. Pardo M, et al. (2010) An expanded Oct4 interaction network: Implications for stem cell biology, development, and disease. Cell Stem Cell 6: 382–395. 204. Liang J, et al. (2008) Nanog and Oct4 associate with unique transcriptional repression complexes in embryonic stem cells. Nat Cell Biol 10: 731–739. 205. van den Berg DLC, et al. (2010) An Oct4-centered protein interaction network in embryonic stem cells. Cell Stem Cell 6: 369–381. 206. Ho L, et al. (2009) An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency. Proc Natl Acad Sci USA 106: 5181–5186. 207. Nishimoto M, et al. (2005) Oct-3/4 maintains the proliferative embryonic stem cell state via specific binding to a variant octamer sequence in the regulatory region of the UTF1 locus. Mol Cell Biol 25: 5084–5094. 208. Botquin V, et al. (1998) New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2. Genes Develop 12: 2073–2090. 209. Tokuzawa Y, et al. (2003) Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development. Mol Cell Biol 23: 2699–2708. 210. Nishimoto M, Fukushima A, Okuda A, Muramatsu M. (1999) The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2. Mol Cell Biol 19: 5453–5465. 211. Reményi A, et al. (2003) Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers. Genes Develop 17: 2048–2059. 212. Wang J, et al. (2006) A protein interaction network for pluripotency of embryonic stem cells. Nature 444: 364–368. 213. Lemischka IR. (2010) Hooking up with Oct4. Cell Stem Cell 6: 291–292. 214. Mullin NP, et al. (2008). The pluripotency rheostat Nanog functions as a dimer. Biochem J 411: 227–231. 215. Reik W. (2007) Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447: 425–432.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
84
10/12/2010
4:38 PM
Page 84
K. M. Loh et al.
216. Yamanaka S, Blau HM. (2010) Nuclear reprogramming to a pluripotent state by three approaches. Nature 465: 704–712. 217. Aasen T, et al. (2008) Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol 26: 1276–1284. 218. Eminli S, et al. (2009) Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells. Nat Genet 41: 968–976. 219. Wernig M, et al. (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448: 318–324. 220. Okita K, Ichisaka T, Yamanaka S. (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448: 313–317. 221. Chin MH, et al. (2009) Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. Cell Stem Cell 5: 111–123. 222. Guenther MG, et al. (2010) Chromatin structure and gene expression programs of human embryonic and induced pluripotent stem cells. Cell Stem Cell 7: 249–257. 223. Stadtfeld M, et al. (2010) Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 465: 175–181. 224. Zhao X-Y, et al. (2009) iPS cells produce viable mice through tetraploid complementation. Nat 461: 86–90. 225. Aasen T, Belmonte JCI. (2010) Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells. Nat Proto 5: 371–382. 226. Yamanaka S. (2007) Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 1: 39–49. 227. Chan EM, et al. (2009) Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat Biotechnol 27: 1033–1037. 228. Stadtfeld M, Maherali N, Breault DT, Hochedlinger K. (2008) Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2: 230–240. 229. Mikkelsen TS, et al. (2008) Dissecting direct reprogramming through integrative genomic analysis. Nature 454: 49–55. 230. Li H, et al. (2009) The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 460: 1136–1139. 231. Utikal J, et al. (2009) Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature 460: 1145–1148. 232. Samavarchi-Tehrani P, et al. (2010) Functional genomics reveals a BMPdriven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7: 64–77. 233. Li R, et al. (2010) A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 7: 51–63.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
85
10/12/2010
4:38 PM
Page 85
Molecular Principles Underlying Pluripotency and Differentiation
234. Silva J, et al. (2008) Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol 6: e253. 235. Ichida JK, et al. (2009) A small-molecule inhibitor of tgf-β signaling replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell 5: 491–503. 236. Sridharan R, et al. (2009) Role of the murine reprogramming factors in the induction of pluripotency. Cell 136: 364–377. 237. Han J, et al. (2010) Tbx3 improves the germ-line competency of induced pluripotent stem cells. Nature 463: 1096–1100. 238. Nakagawa M, Takizawa N, Narita M, et al. (2010) Promotion of direct reprogramming by transformation-deficient Myc. Proc Natl Acad Sci USA. 239. Boiani M, Eckardt S, Schöler HR, McLaughlin KJ. (2002) Oct4 distribution and level in mouse clones: Consequences for pluripotency. Genes Develop 16: 1209–1219. 240. Ramalho-Santos M. (2009) iPS cells: Insights into basic biology. Cell 138: 616–618. 241. Chambers I, et al. (2007) Nanog safeguards pluripotency and mediates germline development. Nature 450: 1230–1234. 242. Nakagawa M, et al. (2008) Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26: 101–106. 243. Wernig M, Meissner A, Cassady JP, Jaenisch R. (2008) c-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell 2: 10–12. 244. Heng J-CD, et al. (2010) The nuclear receptor Nr5a2 can replace Oct4 in the reprogramming of murine somatic cells to pluripotent cells. Cell Stem Cell 6: 167–174. 245. Ichida JK, et al. (2009). A small-molecule inhibitor of tgf-β signaling replaces Sox2 in reprogramming by inducing Nanog. Cell Stem Cell 5: 1–13. 246. Feng B, et al. (2009) Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb. Nat Cell Biol 11: 197–203. 247. Kim JB, et al. (2009) Oct4-induced pluripotency in adult neural stem cells. Cell 136: 411–419. 248. Hanna J, et al. (2008) Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133: 250–264. 249. Hacein-Bey-Abina S, et al. (2003) LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302: 415–419. 250. Nusse R, Varmus HE. (1982) Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell 31: 99–109. 251. Feng B, Ng J-H, Heng J-CD, Ng H-H. (2009) Molecules that promote or enhance reprogramming of somatic cells to induced pluripotent stem cells. Cell Stem Cell 4: 301–312.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
b899_Chapter-02.qxd
86
10/12/2010
4:38 PM
Page 86
K. M. Loh et al.
252. Stadtfeld M, Nagaya M, Utikal J, et al. (2008) Induced pluripotent stem cells generated without viral integration. Science 322: 945–949. 253. Soldner F, et al. (2009). Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136: 964–977. 254. Woltjen K, et al. (2009) piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458: 766–770. 255. Okita K, Nakagawa M, Hyenjong H, et al. (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science 322: 949–953. 256. Stadtfeld M, Hochedlinger K. (2009) Without a trace? Piggy Bacing toward pluripotency. Nat Met 6: 329–330. 257. Stockwell BR. (2000) Chemical genetics: Ligand-based discovery of gene function. Nat Revi Genet 1: 116–125. 258. Maherali N, Hochedlinger K. (2009) Tgfβ signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc. Curr Biol 19: 1718–1723. 259. Shi Y, et al. (2008) Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 3: 568–574. 260. Li W, et al. (2009) Generation of human-induced pluripotent stem cells in the absence of exogenous Sox2. Stem Cells 27: 2992–3000. 261. Lyssiotis CA, et al. (2009) Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4. Proc Natl Acad Sci USA 106: 8912–8917. 262. Huangfu D, et al. (2008) Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat Biotechnol 26: 1269–1275. 263. Zarzeczny A, et al. (2009) iPS cells: mapping the policy issues. Cell 139: 1032–1037. 264. Ying Q-L, et al. (2008) The ground state of embryonic stem cell selfrenewal. Nature 453: 519–523. 265. Izumikawa M, et al. (2005) Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. Nat Med 11: 271–276. 266. Zhou Q, Brown J, Kanarek A, et al. (2008) In vivo reprogramming of adult pancreatic exocrine cells to β-cells. Nature 455: 627–632. 267. Graf T, Enver T. (2009) Forcing cells to change lineages. Nature 462: 587–594.
STEM CELLS - From Bench to Bedside (Second Edition) © World Scientific Publishing Co. Pte. Ltd. http://www.worldscibooks.com/lifesci/7504.html
![[8] Development of Hematopoietic Repopulating Cells from Embryonic Stem Cells](https://kipdf.com/img/300x300/8-development-of-hematopoietic-repopulating-cells-_5ac2fdd21723dd39f0132896.jpg)
![[8] Gene Trapping in Embryonic Stem Cells](https://kipdf.com/img/300x300/8-gene-trapping-in-embryonic-stem-cells_5aae1cdc1723dd1fd76a74dd.jpg)