The many faces of Pluripotency: in vitro adaptations of a continuum of in vivo states

Abstract

Pluripotency defines the propensity of a cell to differentiate into, and generate, all somatic, as well as germ cells. The epiblast of the early mammalian embryo is the founder population of all germ layer derivatives and thus represents the bona fide in vivo pluripotent cell population. The so-called pluripotent state spans several days of development and is lost during gastrulation as epiblast cells make fate decisions towards a mesoderm, endoderm or ectoderm identity. It is now widely recognized that the features of the pluripotent population evolve as development proceeds from the pre- to post-implantation period, marked by distinct transcriptional and epigenetic signatures. During this period of time epiblast cells mature through a continuum of pluripotent states with unique properties. Aspects of this pluripotent continuum can be captured in vitro in the form of stable pluripotent stem cell types. In this review we discuss the continuum of pluripotency existing within the mammalian embryo, using the mouse as a model, and the cognate stem cell types that can be derived and propagated in vitro. Furthermore, we speculate on embryonic stage-specific characteristics that could be utilized to identify novel, developmentally relevant, pluripotent states.

Keywords: Chimaera; Embryonic stem cells; Epiblast stem cells; Epiblast-like cells; Formative; Ground state; Intermediate; Naïve; Pluripotency; Primed.

Fig. 1

A schematic diagram depicting the relationship between in vitro and in vivo pluripotent state progression. The diagram depicts the location of pluripotent cells (red) within the developing mouse embryo from embryonic day (E) 3.5 to E7.5. Prior to E3.5, cells of the pre-implantation embryo are ‘totipotent’, capable of generating both embryonic and extraembryonic cell types. At E3.5, cells in the ICM of the blastocyst are a heterogeneous mix of epiblast (Epi) and primitive endoderm (PrE) precursors. Epi cells are pluripotent and will generate all cells of the embryo-proper, including the germ cells, proper while PrE cells will generate extraembryonic cell types such as the yolk sac. The outer trophectoderm (TE) cells will generate extraembryonic cell types including the fetal portion of the placenta. One day later, at E4.5, the Epi and PrE cells are specified and become physically segregated into two distinct layers and the embryo implants into the uterus. At early post-implantation stages (E5.5) the Epi is in an entirely undifferentiated pluripotent state. At E6.5, cells within the proximal posterior of the embryo are exposed to differentiation-promoting signals from both embryonic and extraembryonic lineages that stimulate the onset of gastrulation and differentiation of cells as they enter the primitive streak (PS) region (yellow). By E7.5, the PS has extended distally and PS derivatives including extraembryonic mesoderm, embryonic mesoderm and definitive endoderm are being generated. The anterior Epi has also started to differentiate into anterior neurectoderm (NE). Pluripotency is lost at approximately E8.0. Pluripotent stem cell lines can be maintained in vitro and appear to resemble various embryonic stages of pluripotency. While embryonic stem cells (ESCs) can be derived from embryos from E3.5 and E7.5 and epiblast stem cells (EpiSCs) can be derived from embryos between E3.5 and E8.0, ESCs resemble the naïve pluripotent state (blue) existing in the early pre-implantation embryo while EpiSCs resemble primed pluripotent cells (green) of the late post-implantation Epi during gastrulation. Intermediate or formative states of pluripotency (orange), between the naïve and primed states likely exist in the embryo. While a number of potential states have been isolated, Epi-like cells (EpiLCs), generated from ESCs in vitro, have been most clearly defined in relation to the embryo and are more transcriptionally similar to E5.75 Epi than EpiSCs are. Representative brightfield images of ESC, EpiLC and EpiSC cultures are shown. Extraembryonic lineages are depicted in gray; dark gray lineages are TE-derived and light gray lineages PrE-derived. A = anterior, P = posterior, Pr = proximal, D = distal

Fig. 2

The role of signaling pathways in self-renewal and differentiation of in vitro pluripotent stem cell populations. Embryonic stem cells (ESCs) represent a naïve state of pluripotency similar to the pre-implantation epiblast (Epi). ESCs are routinely maintained in a self-renewing state in serum (a source of BMP) and LIF (SL). Under these conditions, ESC cultures are heterogeneous and contain subpopulations of lineage-primed cells (yellow and green cells) i.e. cells that coexpress germ layer markers alongside pluripotency markers, and are biased in differentiation towards particular lineages. A more homogeneous ESC state can be generated by blocking FGF signaling using a MEK inhibitor (PD0325901), and activating Wnt signaling using a GSK3 inhibitor (CHIR99021), a condition known as ‘2i’. In these conditions, self-renewal occurs in the absence of external signals, although cell propagation is enhanced in the presence of Wnt pathway activity through CHIR99021. This state is referred to as the naïve or “ground state” of pluripotency. ESCs can be pushed further along the differentiation trajectory by culturing in the presence of FGF and Activin (FA) for 48 hours to generate a cell state referred to as Epi-like cells (EpiLCs). This is a transient cell state, and it is unknown whether self-renewing EpiLCs can be captured by the addition of other factors. It is also not known whether EpiLCs are a homogeneous population of cells. Upon further differentiation in FA over multiple passages, cells resemble a primed state of pluripotency akin to the later post-implantation Epi, referred to as epiblast stem cells (EpiSCs). While FA promotes differentiation of ESCs and EpiLCs, it promotes EpiSC self-renewal. EpiSCs can be derived from ESCs in culture or directly from embryos with FA. When grown in FA, EpiSCs, like ESCs in SL, are heterogeneous and contain lineage-primed populations. While activation of Wnt signaling promotes a naïve ground state of self-renewal, inhibition of Wnt signaling promotes a more homogeneous primed ground state of self-renewal. Therefore cells in naïve and primed pluripotent states respond to signaling factors with opposite outcomes, Wnt and BMP promote self-renewal of the naïve state but differentiation of the primed state of pluripotency and conversely FA promote differentiation of the naïve state but self-renewal of the primed state of pluripotency. Addition of BMP4 and WNT3A in combination with FA stimulates further differentiation of EpiSCs into PS-derived mesoderm and endoderm, while in the absence of FA, BMP or Wnt EpiSCs differentiate to neurectoderm [127]. EpiLCs are the only pluripotent state that has been shown to efficiently generate primordial germ cell-like cells (PGCLCs). Presumably, ESCs have not yet acquired this capacity, while EpiSCs have lost it. Cells within the dashed box are within the pluripotency spectrum while cells outside have differentiated. Blue arrows indicate self-renewal. Orange arrows denote the direction of differentiation along the developmental trajectory

Fig. 3

Different pluripotent states have distinct expression profiles. a. Schematic diagram illustrating the change in relative protein expression levels of the pluripotency-associated genes, NANOG, KLF4 and OCT4 during the transition from a naïve to a primed state of pluripotency. KLF4 is lost as cells exit the naïve state of pluripotency, NANOG is transiently downregulated and OCT4 is maintained at similar levels throughout this period. b. Schematic diagram showing the expression domains of NANOG, KLF4 and OCT4 from embryonic day (E) 3.5 to 7.5 of development. NANOG, KLF4 and OCT4 are all expressed within the ICM of the early blastocyst. While OCT4 is relatively homogeneous, KLF4 and NANOG are both heterogeneously expressed. At E4.5, the epiblast (Epi) homogeneously expresses all 3 of these markers, while the primitive endoderm expresses low levels of OCT4 and KLF4 but not NANOG. At early implantation (E5.5), KLF4 expression is lost and OCT4 and NANOG are coexpressed throughout the Epi. By E6.5–7.5, OCT4 continues to be expressed throughout the Epi while NANOG is restricted to the posterior Epi. c. Representative confocal optical sections of ESC, EpiLC and EpiSC cultures. All cell lines were derived from the 129/Ola E14 parental ESC line. ESCs were maintained in serum and LIF and expressed OCT4, NANOG and KLF4 heterogeneously. EpiLCs expressed OCT4, but downregulated NANOG, and lost KLF4 expression. EpiSCs (derived by culture of E14 ESCs in FGF and Activin for >20 passages) expressed high levels of NANOG and OCT4, but no KLF4