Transcription factor heterogeneity and epiblast pluripotency

Abstract

Stem cells are defined by the simultaneous possession of the seemingly incongruent properties of self-renewal and multi-lineage differentiation potential. To maintain a stem cell population, these opposing forces must be balanced. Transcription factors that function to direct pluripotent cell identity are not all equally distributed throughout the pluripotent cell population. While Oct4 levels are relatively homogeneous, other transcription factors, such as Nanog, are more heterogeneously expressed. Moreover, Oct4 positive cells fluctuate between states of high Nanog expression associated with a high probability of self-renewal and low Nanog expression associated with an increased propensity to differentiate. As embryonic stem (ES) cells transit to the more developmentally advanced epiblast stem cell (EpiSC) state, the levels of pluripotency transcription factors are modulated. Such modulations are blunted in cells that overexpress Nanog and this may underlie the resistance of Nanog-overexpressing cells to transit to an EpiSC state. Interestingly, increasing the levels of Nanog in EpiSC can facilitate reversion to the ES cell state. Together these observations suggest that Nanog lies close to the top of the hierarchy of pluripotent transcription factor regulation.

Figure 1.

Model depicting the stem cell paradox. The cells can either self-renew or differentiate. Populations of stem cells maintain a balance between these opposing forces. This balance is influenced by environmental cues, as well as intrinsic fate regulators. Here, the effect of modulating the expression of the transcription factor Nanog is depicted. In cells that overexpress Nanog, population behaviour is skewed towards self-renewal. In contrast, in Nanog-null cells, the balance is tipped in the opposite direction towards differentiation.

Figure 2.

Transition from naive to primed pluripotency. (a) The developmental changes that occur between the nascent epiblast found in the blastocyst and the post-implantation epiblast can be recapitulated in vitro by changing growth factor supplements to the medium. (b) Gene expression analysis associated with the developmental restriction of pluripotency. Quantitative real-time polymerase chain reaction (qPCR) showing the changes that occur to transcription factor mRNAs when ES cells differentiate into EpiSCs. ES cells were plated on conventional media (LIF/FCS). Twenty-four hours later, the media was switched to activin/FGF. qPCR analyses were determined relative to TATA box-binding protein (TBP) and normalized to the ES cell level of 1.0. Yellow bars, day 1; red bars, day 2; blue bars, day 3; maroon bars, day 4; green bars, EpiSCs.

Figure 3.

The effect of altering Nanog expression on the transition of ES cells to EpiSCs. ES cells overexpressing Nanog fivefold (green bars, 5× Nanog; EF4 cells [45]), or twofold (orange bars, 2× Nanog; RCN cells [48]) and a control line with wild-type Nanog levels (black bars, wild-type ES cells; Oct4GiP [54]) were switched into EpiSC media and cultured for eight passages. (a) Nanog-overexpression prevents downregulation of ES cell-specific transcription factors. qPCR showing mRNA levels of ES cell and EpiSC markers. qPCR analyses were determined relative to TBP and normalized to the ES cell level of 1.0. (b) Cell lines that overexpress Nanog retain the ability to self-renew in stringent ES cell culture conditions; after eight passages in EpiSC culture, cells were replanted in ES cell media (supplemented with LIF and 2i [55]) and colonies scored for expression of the ES cell marker alkaline phosphatase (AP). (c) The level of Nanog influences the ability of ES cells to exit the ES cell-specific attractor. This is represented by the depth of the basin in which the cells are located. ES cells with higher Nanog levels can buffer differentiation with greater efficiency and display a bias towards self-renewal.