Signals between cells are critical for embryonic development and for the differentiation of stem cells into various cell types. Identifying the signals that are needed for stem cell differentiation—and understanding how they produce their effect—are key to the overall goal of using stem cells for therapeutic purposes. This proposal focuses on one such signaling system, the Bmp pathway. Bmps, an acronym for Bone morphogenetic proteins, are a family of proteins that function in a variety of processes in embryonic development. They interact with cells through receptors on the cell surface. These interactions cause the receptors to activate proteins known as smads. Following a Bmp signal, Smad proteins enter the nucleus and cause changes in gene expression, which ultimately lead to changes in the identity or behavior of cells. An important function of Bmps is to promote differentiation of embryonic stem cells along certain pathways. Despite the importance of this activity in stem cell biology, very little is known about how it occurs in molecular terms. One idea is that Bmps promote ES cell differentiation by interfering with the function of a complex of proteins known as polycomb. The polycomb complex is though to help to maintain cells in a stem-like state by interfering with the expression of genes that promote cell differentiation. A number of genes that are Bmp targets are also polycomb targets. The major question of this proposal is how the Bmp pathway causes a loss of polycomb-mediated repression and how this effect relates to the differentiation of human embryonic stem cells into different cell and tissue types.
Statement of Benefit to California:
Embryonic stem cells hold great promise in the treatment of human disease. However the lack of basic information about the genetic programs that control the differentiation of these cells limits the extent to which they can be manipulated in culture, and thus their utility. The central aim of this proposal is to help elucidate how, in specific molecular terms, the genetic program controlling “stemness” is turned off and a set of programs controlling differentiation is turned on. The value these studies to the State of California is that they will contribute to the knowledge base that ultimately will enable human ES cells to be used to rescue defective cells in humans.
SYNOPSIS: The project proposes to investigate the interaction of the bone morphogenic protein (BMP) pathway with polycomb complexes. Polycomb represses differentiation through recruitment of chromatin and DNA modification proteins, working with DNA binding proteins such as Oct4 and Nanog to repress differentiation genes and maintain pluripotency. At least some polycomb target genes are also targets for the BMP pathway, including msx2. The PI identified a BMP-responsive element (BRE) for the mouse msx2 gene, localized to a 560 bp region and a minimal 52 bp core consisting of Smad binding sites and a homeodomain binding site, and also YY1 sites. The goal is to use this BRE to drive GFP in hES cells, with or without added BMPs, sort GFP+ cells and evaluate differentiation and epigenetic modifications at the BRE.
In Aim 1, the PI will generate the cell lines, using the 560 bp enhancer, a mutant version, or a minimal 52 bp element with the hsp68 promoter driving GFP. Lentiviral vectors will transduce hES cells and 2 lines for each isolated. In Aim 2, the PI will examine the protein-binding and methylation status of the msx2 promoter in BMP responding and non-responding stem cells.
INNOVATION AND SIGNIFICANCE: The idea of linking the BMP and polycomb pathways is good and likely to be very important in the overall picture of regulating stem cell state and differentiation. Generation of a faithful reporter for BMP signaling could prove very useful. An understanding should lead to strategies that manipulate stem cell differentiation in vitro.
STRENGTHS: The PI is an expert in BMP regulation and defined the key reagent in this proposed study: the short BMP-responsive transcriptional element. The group has the insights to perform the analysis of BMP function. The PI has assembled a strong collaborative team with complementary expertise, especially recruits with hESC experience. The difference in the fate-response between mouse and human stem cells to BMP treatment might indicate the regulatory basis for this difference. The dose-response experiment alone may produce interesting information.
WEAKNESSES: The PI justifies the project primarily based on possible distinctions between the BMP pathway in mouse and human ES cells. Thus, in mESCs, BMPs promote the stem cell state while, in hESCs, BMPs promote differentiation. Yet, the model suggested is really the same - that BMPs will activate the msx promoter via the identified conserved BRE, leading to recruitment of chromatin and DNA modifiers. Presumably, adding BMPs to the culture will activate the reporter, lead to expression of GFP and msx2, along with the corresponding expected changes in chromatin and DNA, and differentiation. It is therefore not clear why these experiments cannot be done in a much more productive manner in mESCs, and what specifically will be gained by using the hESC model. Since the pathway and the element are so well conserved (justification for its importance), why should it behave differently in hESCs, and if it does, what does that say about the basic premise of the hypothesis in the first place? There is concern that not much new is going to be learned from the effort.
The experiments are likely to confirm changes in chromatin in response to BMP stimulation, but do not include specific tests of mechanisms. It is not clear how the results will inform studies important to the use of human stem cells for therapy, for example to help develop strategies to direct differentiation of hES cells or control self-renewal.
DISCUSSION: There was no further discussion following the reviewers' comments.