Funding opportunities

Epigenetic pre-programming of human ES cells that leads to differentiation bias

Funding Type: 
Basic Biology I
Grant Number: 
Funds requested: 
$1 382 400
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
Human embryonic stem cells (hESCs) carry great potential for cell replacement therapies, since they can differentiate into number of different cell types. Therefore, it is crucial to understand the differentiation and proliferation of hESCs. We previously demonstrated that not all hESCs were created equal by documenting that two NIH hESC lines (i.e., HSF1 and HSF6) generated human neurons with different characteristics when they were differentiated into neural lineages under the same differentiation procedure. Specifically, HSF1 generates cells of forebrain origin while HSF6 gives rise to more posterior and non-forebrain neurons. These findings clearly indicate a differentiation bias between hESCs. Our knowledge on the control of cell-fate specification has been based on studies using animal models. However, not only the human genome is different from those of animals, but also the epigenetic blueprints differ significantly between human and animal stem cells. The epigenetic blueprints determine the specificity, timing, or quantity of expression of particular genes or cohorts of genes. In this proposal, we will reveal the unique epigenetic blueprints of several hESC lines that define their differentiation bias when they are allowed to differentiate along the neural lineage. Our results will reveal the link between the epigenetic blueprint and the differentiation bias of hESCs or induced pluripotent stem (iPS) cells. The approach of this proposal will enable us to predict the differentiation bias of a particular hESC or iPS cell line in the future based on its epigenetic blueprint without laboriously going through differentiation procedures for each lineage to empirically determine whether a particular line can be differentiated into a particular cell lineage. This approach will save tremendous amount of time and resources when the cells are planned to be used for therapeutic applications. Furthermore, our proposed study will reveal whether we can rewrite the epigenetic blueprint in hESCs or iPS cells using number of different factors. Taken together, understanding the genetic and epigenetic mechanisms underlying hESC differentiation bias may shed light on the nature of the developmental program and suggest strategies for controlling hESC and iPS cell differentiation.
Statement of Benefit to California: 
Human embryonic stem cells (hESCs) hold great potential for cell replacement therapy where cells are lost due to disease or injury. For the diseases of the central nervous system, hESC-derived neurons could be used for repair. This approach requires careful characterization of hESCs prior to utilizing their therapeutic potentials. Our preliminary findings demonstrated that certain hESC lines show differentiation bias when they are allowed to differentiate along the neural lineage in culture. In our application, we propose to reveal the unique epigenetic blueprints of several hESC lines that define the differentiation decision of hESCs. The unique epigenetic blueprints may arise from embryo-specific genetic variations or stochastic events accumulated during in vitro culturing. Such a problem, though initially surfaced when dealing with different hESC lines, might also apply to pluripotent stem cells that are derived using the induced pluripotent stem (iPS) cell technology. Our proposed studies will help us understand the genetic and epigenetic mechanisms underlying hESC differentiation bias and it may shed light on the nature of the developmental program controlling hESC and iPS cell differentiation. Numerous residents of California suffer from diseases that could potentially be cured by using stem cell based therapies. In order to be able to use hESCs and/or iPS cells for therapy, better understanding of their differentiation is absolutely needed. There is no question that the information obtained from the experiments of this proposal will benefit the residents of California with respect to stem cell therapy.
Review Summary: 
This proposal is based on the observation that although different human embryonic stem cell (hESC) lines express similar sets of pluripotency genes, they often possess different differentiation biases. A prime example is the comparison between HSF1 and HSF6 cell lines differentiated into neural lineages: the former gives rise to cells with a forebrain phenotype while the latter gives rise to cells with a more caudal, non-forebrain phenotype. The major goals of this proposal are to identify epigenetic signatures in hESC lines with propensities for generating neural cells that correspond to different anterior-posterior levels in vivo, and to develop methods for modifying these signatures. In Aim 1 the applicant proposes to compare epigenetic and gene expression profiles of HSF1 and HSF6 at 3 stages: undifferentiated cells, neural precursor cells, and differentiated neurons. These analyses will also be performed with Noggin and retinoic acid, factors critical for patterning during development. In Aim 2 the applicant will extend the studies described in Aim 1 to the H1 and H9 hESC lines. Finally, in Aim 3, the applicant will manipulate the epigenetic signatures in HSF1 and HSF6 cell lines in an attempt to alter their differentiation bias. Reviewers agreed that this proposal addresses an important scientific question. The identification of epigenetic blueprints for different hESC lines may explain their differentiation biases and could be used to predict the best cell line choice for a particular application. If the applicant is able to modify the epigenetic blueprint, as proposed in Aim 3, this could also provide a significant advantage for future therapeutic applications. Reviewers raised a number of issues with the research plan that caused them to doubt its feasibility. Some of the preliminary data were hard to interpret due to a lack of background information or unclear and incomplete figure legends. This lack of attention to detail was also evident in the paucity of references cited in the proposal. One reviewer felt that the application reflected a general misunderstanding or misinterpretation of the literature surrounding the molecular basis of neural induction and patterning during embryogenesis. This reviewer noted that the use of Noggin as a rostralizing agent is at odds with embryological data. The reviewer wondered whether the applicant has considered alternate explanations for the reported increase in the number of forebrain neurons following Noggin treatment, such as cell line bias. Another reviewer found it surprising that there is no mention of using single-cell derived clonal cell lines for these experiments. This reviewer cautioned that there may be genetic and epigenetic drift over time in culture and suggested analyzing clonal populations derived from single cells at defined passages. With regard to Aim 3, one reviewer questioned its feasibility, given the lack of preliminary data supporting its approach. Finally, reviewers would have appreciated a more thorough discussion of potential pitfalls and alternative approaches. Reviewers agreed that the applicant is a talented junior investigator who has made important contributions to the field. They described the applicant’s publication record as limited but high-impact. In general, the reviewers found the assembled research team to be well-qualified to carry out the work proposed, although one reviewer suggested that it may be helpful to have one individual devote 100% effort to the project. Overall, while the reviewers appreciated the significance of the scientific question addressed in this proposal, the research plan did not inspire confidence in the project’s likelihood of success.

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