The origin of pluripotency

Pluripotency is the capacity of a single cell to form all somatic and germline cells of the organism in a regulated manner steered by extrinsic cues. A pluripotent cell can be considered as a blank slate. My research is centred on understanding how this tabula rasa is created, how it can be maintained, and how it acquires direction.

The mammalian zygote is programmed to produce extra-embryonic trophoblast cells. Therefore pluripotent cells must be generated de novo. Pluripotency develops within the inner cell mass (ICM) of the pre-implantation blastocyst. Our laboratory and others have shown that emergence of the pluripotent state is orchestrated by two transcriptional organisers, Oct4 and Nanog. These factors confer on a subset of ICM cells, the epiblast, potency to generate embryonic lineages. Naive epiblast cells persist only briefly in the embryo. However, they may be immortalised in vitro as embryonic stem (ES) cells.

Simplifying stem cell culture

A strategic goal of our research has been to simplify the complex culture environment originally employed for ES cells so as to define the essential requirements for maintaining pluripotency. We found that co-culture could be replaced by the cytokine leukaemia inhibitory factor (LIF) which activates the transcription factor Stat3 and feeds into the core pluripotency network. More recently, realisation that ES cell commitment is triggered by the mitogen activated protein kinase (MAPK) cascade has allowed replacement of serum with highly selective small molecule inhibitors. Suppression of MAPK maximises ES cell self-renewal in the presence of LIF or alternatively combined with partial inhibition of glycogen synthase kinase-3, which stabilises β-catenin.

The ground state of pluripotency

The ability to withstand inhibition of MAPK signalling is unusual for non-transformed cells. We have proposed that ES cells represent a ground state for mammalian cells, meaning a basal self-replicating state that is developmentally and epigenetically unprogrammed and has minimal requirements for extrinsic stimuli to support survival and proliferation. This ground state is highly sensitive to perturbation by MAPK. We hypothesise that the underlying molecular network may be conserved among eutherian mammals. Indeed MAPK inhibition enables derivation of ES cells from all strains of mice tested and also from rats, which was not previously possible. Moreover, blockade of MAPK in the developing mouse embryo reveals that the entire ICM has the potential to acquire pluripotency. These finding lead to a reinterpretation of the nature of ES cells, suggesting that they are most likely identical to early epiblast cells in the blastocyst rather than an artefact of in vitro manipulation. The next challenge is to elucidate how pluripotent cells exit from the ground state and select somatic lineage or germline differentiation paths.

From rodents to mankind

Inhibition of MAPK signalling also facilitates the generation of induced pluripotent stem (iPS) cells by molecular reprogramming. However, so-called human “ES” cells and human iPS cells are not sustained by LIF or MAPK inhibition. On the contrary, they depend on MAPK signalling for continued proliferation. A possible explanation for this divergence is that the currently available human cells may be representative of a later stage of development. A critical unanswered question therefore is whether a ground state equivalent to rodent ES cells exists for all mammals? We are investigating this issue using donated supernumerary human embryos. The variable quality of material available and the lack of functional measures of blastocyst maturation make this research problematic, however. We will therefore also examine development of pluripotency and establishment of ES cells in other mammalian embryos.

Towards regenerative medicine

Our work is of a very fundamental nature. Nonetheless, by contributing to the understanding of the mechanisms leading to the self-renewal of embryonic stem cells and by defining the culture environment for these cells, our research points the way for biomedical applications of stem cells. These studies are a vital step for the advancement of regenerative medicine, where stem cells could in future be used to regenerate damaged organs or tissues and for the treatment of various diseases.

Figure 1 :
The core transcription factor circuitry or pluripotency
When differentiation stimuli are inhibited the intrinsic transcription factor circuitry is sufficient to sustain efficient ES cell self-renewal without necessity for LIF/Stat3. Krüppel-like factors, in particular Klf2 which is a direct target of Oct4, may be key components of the network. If differentiation is enabled, activation of Stat3 becomes essential. We hypothesise that Stat3 acts by reinforcing the core circuitry. One way in which this is achieved is by inducing Krüppel-like factors Klf4 and Klf5, thereby increasing total Klfs above the basal levels directed by Oct4/Sox2.

Figure 2 :
The ground state transition to lineage differentiation
Pluripotent ES cells self-renew in the presence of LIF or are triggered to enter into differentiation by activation of mitogen activated protein kinase (MAPK). We surmise that unopposed MAPK signalling induces changes in protein activity and gene expression that destabilise the pluripotent circuitry. Nanog may resist or revert such perturbation; otherwise cells reach a critical state that resolves via commitment and lineage restriction.

Professeur Austin Smith
Medical Research Council Professor &
Director of the Wellcome Trust Centre for Stem Cell Research
University of Cambridge
Tennis Court Road
UK-CAMBRIDGE, CB2 1QR

Tél.: + 44 (0)1223 760 233
Fax: + 44 (0)1223 760 241

austin.smith@cscr.cam.ac.uk
Assistante agssec@cscr.cam.ac.uk

www.cscr.cam.ac.uk/research/smith/smith.html
www.eurosystemproject.eu EuroSyStem Project
www.eurostemcell.org European Portal for Stem Cell Research (EuroStemCell)