Austin Smith

Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells

Cell fate during development is defined by transcription factors that act as molecular switches to activate or repress specific gene expression programmes. The POU transcription factor Oct-3/4 (encoded by Pou5f1) is a candidate regulator in pluripotent and germline cells and is essential for the initial formation of a pluripotent founder cell population in the mammalian embryo. Here we use conditional expression and repression in embryonic stem (ES) cells to determine requirements for Oct-3/4 in the maintenance of developmental potency. Although transcriptional determination has usually been considered as a binary on-off control system, we found that the precise level of Oct-3/4 governs three distinct fates of ES cells. A less than twofold increase in expression causes differentiation into primitive endoderm and mesoderm. In contrast, repression of Oct-3/4 induces loss of pluripotency and dedifferentiation to trophectoderm. Thus a critical amount of Oct-3/4 is required to sustain stem-cell self-renewal, and up- or downregulation induce divergent developmental programmes. Our findings establish a role for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and illustrate the sophistication of critical transcriptional regulators and the consequent importance of quantitative analyses.

Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4

Oct4 is a mammalian POU transcription factor expressed by early embryo cells and germ cells. We report that the activity of Oct4 is essential for the identity of the pluripotential founder cell population in the mammalian embryo. Oct4-deficient embryos develop to the blastocyst stage, but the inner cell mass cells are not pluripotent. Instead, they are restricted to differentiation along the extraembryonic trophoblast lineage. Furthermore, in the absence of a true inner cell mass, trophoblast proliferation is not maintained in Oct4-/- embryos. Expansion of trophoblast precursors is restored, however, by an Oct4 target gene product, fibroblast growth factor-4. Therefore, Oct4 also determines paracrine growth factor signaling from stem cells to the trophectoderm.

FUNCTIONAL EXPRESSION CLONING OF NANOG, A PLURIPOTENCY SUSTAINING FACTOR IN EMBRYONIC STEM CELLS

Embryonic stem (ES) cells undergo extended proliferation while remaining poised for multilineage differentiation. A unique network of transcription factors may characterize self-renewal and simultaneously suppress differentiation. We applied expression cloning in mouse ES cells to isolate a self-renewal determinant. Nanog is a divergent homeodomain protein that directs propagation of undifferentiated ES cells. Nanog mRNA is present in pluripotent mouse and human cell lines, and absent from differentiated cells. In preimplantation embryos, Nanog is restricted to founder cells from which ES cells can be derived. Endogenous Nanog acts in parallel with cytokine stimulation of Stat3 to drive ES cell self-renewal. Elevated Nanog expression from transgene constructs is sufficient for clonal expansion of ES cells, bypassing Stat3 and maintaining Oct4 levels. Cytokine dependence, multilineage differentiation, and embryo colonization capacity are fully restored upon transgene excision. These findings establish a central role for Nanog in the transcription factor hierarchy that defines ES cell identity.

The ground state of embryonic stem cell self-renewal.

In the three decades since pluripotent mouse embryonic stem (ES) cells were first described they have been derived and maintained by using various empirical combinations of feeder cells, conditioned media, cytokines, growth factors, hormones, fetal calf serum, and serum extracts. Consequently ES-cell self-renewal is generally considered to be dependent on multifactorial stimulation of dedicated transcriptional circuitries, pre-eminent among which is the activation of STAT3 by cytokines (ref. 8). Here we show, however, that extrinsic stimuli are dispensable for the derivation, propagation and pluripotency of ES cells. Self-renewal is enabled by the elimination of differentiation-inducing signalling from mitogen-activated protein kinase. Additional inhibition of glycogen synthase kinase 3 consolidates biosynthetic capacity and suppresses residual differentiation. Complete bypass of cytokine signalling is confirmed by isolating ES cells genetically devoid of STAT3. These findings reveal that ES cells have an innate programme for self-replication that does not require extrinsic instruction. This property may account for their latent tumorigenicity. The delineation of minimal requirements for self-renewal now provides a defined platform for the precise description and dissection of the pluripotent state.

BMP Induction of Id Proteins Suppresses Differentiation and Sustains Embryonic Stem Cell Self-Renewal in Collaboration with STAT3

The cytokine leukemia inhibitory factor (LIF) drives self-renewal of mouse embryonic stem (ES) cells by activating the transcription factor STAT3. In serum-free cultures, however, LIF is insufficient to block neural differentiation and maintain pluripotency. Here, we report that bone morphogenetic proteins (BMPs) act in combination with LIF to sustain self-renewal and preserve multilineage differentiation, chimera colonization, and germline transmission properties. ES cells can be propagated from single cells and derived de novo without serum or feeders using LIF plus BMP. The critical contribution of BMP is to induce expression of Id genes via the Smad pathway. Forced expression of Id liberates ES cells from BMP or serum dependence and allows self-renewal in LIF alone. Upon LIF withdrawal, Id-expressing ES cells differentiate but do not give rise to neural lineages. We conclude that blockade of lineage-specific transcription factors by Id proteins enables the self-renewal response to LIF/STAT3.

Changing potency by spontaneous fusion

Recent reports have suggested that mammalian stem cells residing in one tissue may have the capacity to produce differentiated cell types for other tissues and organs1–9. Here we define a mechanism by which progenitor cells of the central nervous system can give rise to non-neural derivatives. Cells taken from mouse brain were co-cultured with pluripotent embryonic stem cells. Following selection for a transgenic marker carried only by the brain cells, undifferentiated stem cells are recovered in which the brain cell genome has undergone epigenetic reprogramming. However, these cells also carry a transgenic marker and chromosomes derived from the embryonic stem cells. Therefore the altered phenotype does not arise by direct conversion of brain to embryonic stem cell but rather through spontaneous generation of hybrid cells. The tetraploid hybrids exhibit full pluripotent character, including multilineage contribution to chimaeras. We propose that transdetermination consequent to cell fusion10 could underlie many observations otherwise attributed to an intrinsic plasticity of tissue stem cells9.

Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture

Mouse embryonic stem (ES) cells are competent for production of all fetal and adult cell types. However, the utility of ES cells as a developmental model or as a source of defined cell populations for pharmaceutical screening or transplantation is compromised because their differentiation in vitro is poorly controlled. Specification of primary lineages is not understood and consequently differentiation protocols are empirical, yielding variable and heterogeneous outcomes. Here we report that neither multicellular aggregation nor coculture is necessary for ES cells to commit efficiently to a neural fate. In adherent monoculture, elimination of inductive signals for alternative fates is sufficient for ES cells to develop into neural precursors. This process is not a simple default pathway, however, but requires autocrine fibroblast growth factor (FGF). Using flow cytometry quantitation and recording of individual colonies, we establish that the bulk of ES cells undergo neural conversion. The neural precursors can be purified to homogeneity by fluorescence activated cell sorting (FACS) or drug selection. This system provides a platform for defining the molecular machinery of neural commitment and optimizing the efficiency of neuronal and glial cell production from pluripotent mammalian stem cells.

Nanog safeguards pluripotency and mediates germline development

Nanog is a divergent homeodomain protein found in mammalian pluripotent cells and developing germ cells. Deletion of Nanog causes early embryonic lethality, whereas constitutive expression enables autonomous self-renewal of embryonic stem cells. Nanog is accordingly considered a core element of the pluripotent transcriptional network. However, here we report that Nanog fluctuates in mouse embryonic stem cells. Transient downregulation of Nanog appears to predispose cells towards differentiation but does not mark commitment. By genetic deletion we show that, although they are prone to differentiate, embryonic stem cells can self-renew indefinitely in the permanent absence of Nanog. Expanded Nanog null cells colonize embryonic germ layers and exhibit multilineage differentiation both in fetal and adult chimaeras. Although they are also recruited to the germ line, primordial germ cells lacking Nanog fail to mature on reaching the genital ridge. This defect is rescued by repair of the mutant allele. Thus Nanog is dispensible for expression of somatic pluripotency but is specifically required for formation of germ cells. Nanog therefore acts primarily in construction of inner cell mass and germ cell states rather than in the housekeeping machinery of pluripotency. We surmise that Nanog stabilizes embryonic stem cells in culture by resisting or reversing alternative gene expression states.

Naive and Primed Pluripotent States

After maternal predetermination gives way to zygotic regulation, a ground state is established within the mammalian embryo. This tabula rasa for embryogenesis is present only transiently in the preimplantation epiblast. Here, we consider how unrestricted cells are first generated and then prepared for lineage commitment. We propose that two phases of pluripotency can be defined: naive and primed. This distinction extends to pluripotent stem cells derived from embryos or by molecular reprogramming ex vivo.

Nanog Is the Gateway to the Pluripotent Ground State

Summary Pluripotency is generated naturally during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Pluripotency can be recreated by somatic cell reprogramming. Here we present evidence that the homeodomain protein Nanog mediates acquisition of both embryonic and induced pluripotency. Production of pluripotent hybrids by cell fusion is promoted by and dependent on Nanog. In transcription factor-induced molecular reprogramming, Nanog is initially dispensable but becomes essential for dedifferentiated intermediates to transit to ground state pluripotency. In the embryo, Nanog specifically demarcates the nascent epiblast, coincident with the domain of X chromosome reprogramming. Without Nanog, pluripotency does not develop, and the inner cell mass is trapped in a pre-pluripotent, indeterminate state that is ultimately nonviable. These findings suggest that Nanog choreographs synthesis of the naive epiblast ground state in the embryo and that this function is recapitulated in the culmination of somatic cell reprogramming.