Regulation of human embryonic stem cell fate by different forms of the Polycomb transcriptional silencing machinery.
We propose to investigate the mechanisms that maintain embryonic stem cells in the undifferentiated, pluripotent state and how those mechanisms change to allow cells to initiate the first steps of embryonic development. Work in mouse embryonic stem cells suggests that genes that will be turned on during early embryonic development are primed for expression but kept silenced by a complex of regulatory proteins termed the Polycomb group machinery (PcG). We will investigate whether different forms of the PcG machinery components are expressed in human embryonic stem cells compared to differentiating early embryonic cells and whether different forms of the PcG complex bind to and silence different target genes. Our proposed work will uncover fundamental mechanisms that allow human embryonic stem cells to grow and proliferate in the undifferentiated state while maintaining their pluripotent character. These properties are critical for the ability to expand embryonic stem cell populations in order to be able to use the cells for screens for potential therapeutic treatments (if derived from disease carrying individuals). Maintenance of the pluripotent state is also critical for expanding stem cell populations for use in possible cell based transplantation therapies. In addition, our work may reveal mechanisms that help guide early embryonic cells toward particular differentiation pathways, such as neuronal or endoderm or muscle precursors, during the early stages of development. Knowledge of these mechanisms will help in the design of strategies for inducing human embryonic stem cells to initiate differentiation into specific cell types, a key step for cell based regenerative medicine.
Statement of Benefit to California:
The proposed research will lay important groundwork for the ability to grow and guide the differentiation of human embryonic stem cells. These capabilities underlie all ability to use embryonic stem cells for screens for therapies and for embryonic stem cell based regenerative medicine. The proposed experiments may uncover fundamental mechanisms that allow human embryonic stem cells to grow and proliferate in the undifferentiated state while maintaining their pluripotent character. Maintenance of the pluripotent state is critical for expanding stem cell populations for use in possible cell based transplantation therapies. These properties are also critical for the ability to expand embryonic stem cell populations in order to be able to use the cells for screens for potential therapeutic treatments. In addition, the proposed research may reveal mechanisms that help guide early embryonic cells toward particular differentiation pathways, such as neuronal or endoderm (precursor to pancreas and liver) or muscle precursors, during the early stages of development. Knowledge of these mechanisms will help in the design of strategies for inducing human embryonic stem cells to initiate differentiation into specific cell types, a key step for cell based regenerative medicine.
SYNOPSIS: The goal of this proposed research is to examine whether human ESC fate and early development might be regulated by expression of different forms of the Polycomb transcriptional silencing machinery, as occurs during mouse ESC development. The first Aim will determine if Polycomb components are differentially expressed in proliferating versus differentiating human ESCs using hybridization technologies to monitor mRNAs and isoform-specific antibodies to confirm that changes occur at the protein level as well. Aim 2 will examine whether changes in the nature or subnuclear localization of Polycomb components correlates with induction of differentiation toward different germ-layer fates. Aim 3 will use ChIP-on-Chip technology to test whether changes in the subunit composition of the Polycomb silencing machinery components reflect binding to different sets of target genes consistent with the direction of cellular differentiation. SIGNIFICANCE AND INNOVATION: The underlying hypothesis and the experimental plan are well-grounded in recent discoveries about the role of Polycomb proteins in early embryonic cells that must either continue replicating while maintaining pluripotency or begin developmental programs, the decision point critical to strategies to expand ES cell populations or to direct their differentiation to cell types for transplantation therapies. Polycomb components are enriched in stem cells but lowered in the progeny that begin differentiating, different forms of the Polycomb complexes are present during stem versus differentiating phases, and different Polycomb complexes can be localized to different subnuclear compartments consistent with separate functions or sets of target genes. It is important to determine whether these observations with mouse ES cells and Drosophila germ line stem cells also hold for the programmed differentiation of human ES cells and whether this is an important mechanism for controlling ES cell pluripotency. The answers can be expected to be meaningful in the long term for designing strategies to control alternate developmental fates in vivo for the production of clinically useful cells for transplantation therapies. STRENGTHS: Recent discoveries of the role of Polycomb control of mouse ES cell development and the expertise of the PI and her colleagues with the Polycomb machinery of gene silencing, the tissue-specific transcriptional regulatory machinery and the functional interaction between the two place them in a strong position to make meaningful advances in understanding the role of Polycomb in ES cell biology. This work will extend the strong precedents of mouse ES cell discoveries to human ES cells and advance the nature of the studies to the level of protein analysis of multiple Polycomp components and their differentially expressed isoforms. The collaborative assistance from co-PI’s and collaborators Wong, Clarke and Baker at Stanford will bring valuable specialized expertise with ChIP-on-Chip analysis of embryonic stem cells, Polycomb antibodies, and the growth, maintenance and differentiation of new human ESC lines derived and grown in the absence of feeder cells. There can be little doubt that control mechanisms so fundamental as to be conserved from the stem cells of flies to mice will be critical for human stem cell biology as well virtually ensuring the relevance of this proposed investigation. The experimental plan is well designed to answer fundamental questions about universal mechanisms for transcriptional control of ES cell replication while maintaining pluripotency versus the induction of differentiation. The answers can be expected to help design strategies to experimentally control these alternate fates for the production of clinically useful cells for transplantation therapies. This is an excellent investigator with a great track record. The obvious importance of polycomb group proteins is considered a strength. WEAKNESSES: No tests are proposed to confirm function of Polycomb isoforms during differentiation to confirm the implications of correlative evidence obtained. No initial studies are proposed to begin intervention of Polycomb functions to manipulate human stem cells. Technical problems regarding ChIP-on-Chip experiments or potential problems obtaining suitable antibodies will likely be overcome by this excellent research group. The emphasis on the use of embryoid bodies to analyze specific changes of Polycomb proteins during differentiation may be misplaced. The alternative use of proven protocols for directed neural or endodermal differentiation of human ESCs may be more useful to derive maximal value from global analyses like ChIP-on-Chip and microarray hybridization and for long-term goals, especially if feasible for Dr. Baker’s feeder-free cells. The work is all descriptive: are PcG components expressed and do they change in differentiation of hESCs? does the subcellular distribution change? are they associated with different genes? The lab seems well funde, publishes infrequently but in the best journals. Thus much of the work could be done without this funding given the experience and colllaborators mentioned on pg 8 This comes across as a laundry list of preliminary inquiries that will fall short of identifying mechanisms. DISCUSSION: Reviewer 1 stated that s/he was awed by the clarity and precision of the science, but had concerns with the relevance and innovation. Reviewer 2 concurred, saying that this is a good PI with a good lab in a well-established field of study, but the experiments are too descriptive. There was a sense that because Polycomb proteins have survived evolution they are worthy of study but that changes in their expression or localization may not be indicative of a role in differentiation. Since there are no direct tests presented to determine whether the information is applicable to stem cell biology, the proposal falls short of identifying the mechanism or significance of any results.