MMSR

The Molecular Mechanisms of Stem Cell Self Renewal

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 Obiettivo del progetto (Objective)

'Over the past few years, a view has emerged that pluripotency in mouse embryonic stem cells (ESCs) is cell-autonomous and does not depend on external signals. This was based on work that showed that a combination of a small molecule ERK inhibitor with a GSK3 kinase inhibitor was sufficient to maintain self-renewal, in absence of any growth factors. I have now discovered that autocrine or paracrine signalling via the Wnt pathway plays an essential role in ESC self-renewal, and is required to inhibit their differentiation towards primitive ectoderm. Moreover, I have discovered that the combination of Wnt protein with the cytokine LIF is sufficient to support the maintenance and derivation of germ-line competent ES cells in fully defined conditions not containing any other growth factor. Thus, two different fully defined conditions both maintain self-renewal, but do so in different ways. However, the intracellular events downstream of those conditions must both converge on the self-renewal machinery. By analyzing and comparing these events in both systems, unmatched synergy in zooming in on the relevant entrances into the self-renewal machinery can be obtained. I propose to analyze the events downstream of these two sets of self-renewal stimuli, in an effort to identify novel self-renewal genes. The downstream effects of the self-renewal stimuli will be analyzed using gene expression profiling, ChIP-Seq, and phosphoproteomic approaches. Combining and comparing the different data sets will identify candidate genes that will be screened in a self-renewal assay designed for high sensitivity and throughput. Genes that pass this test will be further analyzed for their roles in proliferation, survival, and differentiation in an effort to compartmentalize the self-renewal machinery into functional blocks. This analysis will provide the first comprehensive resource documenting the molecular workings of self-renewal.'

Introduzione (Teaser)

Stem cells are capable of self-renewal and give rise to specialised cell types. EU research has delved into the biochemical cascades involving signalling proteins and stem cells in the early development of germ layers in the embryo.

Descrizione progetto (Article)

Pluripotent stem cells hold great promise in the fields of regenerative medicine and disease modelling. They exist in naive and primed states, the mouse versions being embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs), respectively.

The MMSR (The molecular mechanisms of stem cell self renewal) project has investigated the role of Wnt signalling proteins in the self-renewal and differentiation of human and mouse pluripotent stem cells. Having a high degree of conservation across species, from Drosophila to humans, Wnt signalling lies at the foundation of embryonic development. Moreover, its dysfunction is linked with diseases such as insulin sensitivity and cancer.

MMSR researchers made some surprising discoveries. Wnt signals act as self-renewal factors for ESCs and are indeed required to inhibit their development into EpiSCs. Furthermore, the source of the Wnt proteins is the ESCs themselves and they therefore regulate the naive-to-primed pluripotency transition. As human ESCs (hESCs) differ from mouse ESCs and possess some degree of primed pluripotency, this may be significant in the reprogramming of adult human cells to the pluripotent state.

One of the major obstacles in therapeutic applications of hESCs is that there is limited control of differentiation. The researchers showed that endogenous Wnt signals are hidden mediators of growth factor-induced differentiation, specifically Bmp4. A member of the bone morphogenetic protein family, Bmp4 induces mesoderm formation via gastrulation factors. In contrast, Bmp4 is Wnt independent in the induction of trophoblast differentiation and this renders it useful for differentiation to either lineage.

Further control of differentiation is possible as endogenous Wnt signals interfere with self-renewal of hESCs and mouse EpiSCs. Indeed, Wnt inhibition is so effective in suppressing differentiation that there is no need for the usual manual removal of differentiated cells, greatly reducing the skills and labour involved in working with hESCs.

Project results promise to have an impact on the guided differentiation of hESCs in the areas of stem cell therapy and research. This molecular analysis represents a first as a comprehensive resource of the molecular dynamics of self-renewal.