Stem cells are present in multicellular organisms and are ultimately responsible for generating each of its different cell types. The most 'primitive' stem cells are termed 'totipotent', since they can differentiate into any cell type. As stem cells differentiate and commit to a particular lineage, the range of cells which can subsequently be generated is diminished; pluripotent cells are capable of differentiating into any cells of the three germ layers [Figure 1], while multipotent cells may be further committed, for example, to hematopoietic cell lineages. Isolated stem cells, at any of these varying levels of lineage commitment, can be maintained in their pluripotent state, or be induced to commit to a particular pathway by manipulation of culture media.

Figure 1: Pluripotent stem cell differentiation. Embryonic stem cells (ESC) are derived from the inner cell mass of a blastocyst. They are characterised by their ability to differentiate into all derivative cells types of the three primary germ layers, equating to over 200 different cell types in an adult human. These include the muscle cells (gut and cardiac) of the mesoderm, lung and pancreatic cells of the endoderm, and neuronal and epidermal cells of the ectoderm. Figure taken from http://www.sigmaaldrich.com/life-science/stem-cell-biology.html.

Figure 2: Core transcription factor interactions in the embryonic stem cell (ESC) circuit. The trophectoderm lineage is determined by the antagonism between Oct4 and Cdx2, whereas the balance between Gata-6 and Nanog determines the endoderm lineage. The dashed red line indicates an interaction which emerges out of ChIP-chip data. It is also supported by the phenomenological observation that over-expression of Oct4 ultimately leads to the endoderm lineage, in which Gata-6 is strongly expressed. Figure modified from [2].

Classically, research has focused on isolation of the most primitive of stem cells, on maintaining maximal potency, and on identifying the factors which induce commitment along a particular lineage. More recently, some progress has been made in reprogramming terminally differentiated cells, thereby regressing them back into an embryonic stem cell (ESC) like state [1]. This has established a core set of transcription factors (TFs) that control the stem cell's fate, determining whether it maintains its pluripotency, or whether it commits to a differentiation path.

In this study [2, BIOMD0000000209-BIOMD0000000210 ], the authors construct a dynamical model based upon the minimal regulatory circuit, involving the TFs (Oct4, Sox2 and Nanog) identified [Figure 2], and on available expression data and experimental observations. The model, which builds on previous work [3, BIOMD0000000203-BIOMD0000000204], examines the conditions under which either trophectoderm or endoderm lineage committal occurs, as well as determining the status of the cell under which pluripotency would be sustained.

Key observations which the model must satisfy:

• excess Oct4 over Cdx2 maintains pluripotent stem cell state.
• excess Gata-6 over Nanog gives rise to endoderm lineage cells.
• excess Cdx2 over Oct4 gives rise to trophectoderm lineage cells.

The core TFs determine 3 stable states, and the specific combinations determining lineage commitment, and how the expression levels of each TF are toggled was explored. One observation that could potentially be explained by this model was the biphasic response of Gata-6 as a function of Oct4. The proposed mechanism is that the Oct4-Sox2 (O/S) heterodimer interacts with Gata-6, and this new heterodimer suppresses Nanog. At higher levels of O/S, Gata-6 levels rise, suppressing Nanog and allow Gata-6 to be switched on. These assumptions were converted into ODEs, and the steady state curves for Gata-6 and Nanog with respect to O/S [Figure 3] reflect biphasic behaviour, matching experimental observations.

Figure 3: Steady state curves of Gata-6 and Nanog as a function of input O/SNanog displays a ‘‘bell shaped’’ curve, while Gata-6 displays an ‘‘inverted bell shaped’’ curve. The steady state curves also show two saddle-node bifurcations, indicating a bistable state. The bistability arises due to the cooperative effects between autoregulation of Gata-6 and the repression of Nanog by Gata-6-Oct4. The dotted line indicates the unstable states. Figure taken from [2].

Figure 4: Time series concentrations of Oct4, Sox2, Nanog, Cdx2, Gata-6 and Gcnf. The trophectoderm and endoderm lineages are the only possible states of the system for low and high A. However, for intermediate A, the initial conditions of Oct4/Sox2/Nanog determine the final steady state as can be seen for upper right and lower left, which give either the embryonic or differentiated stem cell (endoderm) lineage, depending on whether the initial conditions were relatively high/low values of [O],[S]&[N]. The system is bistable, and hence can choose either of the two states depending on the initial conditions. Figure taken from [2].

To study the expression levels of TF combinations in the different lineages, an external factor, A, was introduced which activates Oct4 [Figure 4]:

• Trophectoderm state [Figure 4, upper left]:When A is low, Oct4 / Cdx2 balance tips towards Cdx2. This activates Gcnf, which represses Oct4. Since Nanog is also off during this time, Gata-6 is expressed, and remains at high levels through autoregulation.
• Stem cell state [Figure 4, upper right]:At intermediate A, Oct4 is activated, and Oct4, Sox2 and Nanog are switched on. Oct4 suppresses Cdx2, and Nanog suppresses Gata-6, hence there is no negative feedback to Oct4 through unexpressed Gcnf. Interestingly, this region is bistable such that when Oct4, Sox2 and Nanog are set initially to low levels, the system does not reach the stem cell state [Figure 4, lower left].
• Endoderm state [Figure 4, lower right]:At high A, Oct4 level is sufficient to activate Gata-6, suppressing Nanog. The positive feedback to Sox2 and Oct4 from Nanog is therefore removed, and Sox2 levels drop. Cdx2 is suppressed by the high levels of Oct4 (due to A). Oct4 is subjected to some repression through Gcnf, but this is minimal due to high Oct4 expression through A. Gata-6 is also expressed in this state.

Given cells in a particular differentiated state, the perturbations required to shift the lineage back to that of a stem cell can be analysed with this model. For example, endoderm cells display Gata-6, Gcnf and Oct4 expression. To simulate reversion to stem cell state, the authors modified the mathematical expressions for Gata-6 and Nanog by incorporating the external factors SG, to effect the repression of Gata-6, and SN, to induce Nanog expression [Figure 5].

The authors extracted the network components from experimental data and literature mining, and generated a dynamical model. By incorporating 2 basic assumptions, namely that Oct4 activates Gata-6, and Nanog is repressed by O/S-Gata-6, the authors were able to reproduce the biphasic regulation of core TFs that control the committal of stem cells to a differentiated state.

The computational model in this work provides some insight into how core TFs are regulated and the resultant lineages determined. It provides a platform from which cell reprogramming strategies can be explored through the use of directed perturbations.

Figure 5: Left: Steady state concentrations of Oct4, Nanog and Gata-6 as functions of an external signal SG, which represses Gata-6. Although GATA-6 levels decrease, as SG increases, Nanog and Sox2 fail to get induced (unless an external perturbation on Nanog is applied: red arrow) and hence the default embryonic state is not achieved. Right: Steady state concentrations of Oct4, Nanog and Gata-6 as functions of the external signal SN which induces Nanog. Induction of Nanog leads to the reinforcement of the Oct4-Sox2-Nanog sub-system, due to their shared positive feedback regulations: Nanog therefore shuts down Gata-6, and ultimately the embryonic state is attained. Figure taken from [2].