Human embryonic stem (ES) cells are a remarkable cell type that are derived from a group of cells called the inner cell mass (ICM) of a very early stage embryo (about 100 cells in total) obtained from in vitro fertilization program. Human ES cells can be expanded in culture in an undifferentiated state (self-renewal) without limit while retaining the capacity to differentiate into nearly any type of cell. Human ES cells offer an important renewable resource for future cell replacement therapies for many diseases such as Parkinson’s disease, spinal cord injury etc. However, before the full potential of human ES cells can be exploited in the clinic, we need to understand more about human ES cells so we can control their fate towards either self-renewal or towards differentiation into a specific cell type required for cell replacement therapy. Currently it is a problem just to grow human ES cells, let alone to understand how human ES cells make their choice between self-renewal and differentiation. In contrast, several signaling pathways which are important for mouse ES cell self-renewal have been identified, and as a result of this, it is possible to grow mouse ES cells in a fully defined condition. However, these pathways seem to be not operating in human ES cells. This would argue that human ES cells are very different from mouse ES cells, and that understanding of human ES cells may not benefit from the research of mouse ES cells. However, we have recently made striking discoveries on mouse ES cells. We found that for mouse ES cell self-renewal does not require any added growth factors or cytokines but only the elimination of signals that induce differentiation. These new findings provide us with a new prospective to understand human ES cells. Through understanding some of the basic mechanisms involved in human ES cell maintenance, we should be able to develop a more efficient and better method to grow human ES cells, which is clearly important if these cells are to be used clinically.
Human embryonic stem (ES) cells can be maintained indefinitely while retaining the ability to make any type of human tissue. In the future, human ES cells may hold the key to replacing cells lost in many devastating diseases such as Parkinsons and diabetes. But for human ES cells to be of use clinically, they will first have to be multiplied in very large numbers. Scientists must, therefore, learn how to control the growth of stem cells in the laboratory. When a human ES cell divides it can either produce identical copies of itself (self-renewal) or it can produce other more specialised cell types, such as nerve or muscle cells. Understanding how a stem cell makes this choice between self-renewal and differentiation is the central challenge in stem cell research. This proposal is intended to apply the knowledge we obtained from extensive research on mouse ES cells for the better understanding how human ES cells make their decision whether to self-renewal or to differentiate. The direct benefit from this proposal research will be the development of more efficient culture conditions for the growth of human ES cells, which is a critical step leading to the clinical application of human ES cell-derived cells.
The project is focused on understanding the requirements for maintaining stem cell self-renewal activity. While specific distinctions between mouse and human ES are often highlighted (eg. LIF vs FGF) the PI has generated data indicating that the fundamental mechanisms that control self-renewal might not be so different after all. His goal is to define the circuitry that maintains renewal, so that lines can be most efficiently generated and maintained, under defined conditions. His key insight came from showing that mES lines could be generated in the absence of LIF (using Stat3 null mice) as long as MAPK and GSK3 pathways were inhibited (unpublished but submitted). Furthermore, mES cells lacking eRas require FGF for maintenance, while hES cells lack eras activity.
SIGNIFICANCE AND INNOVATION: The application will explore the pathways that are essential for self-renewal of human ES cells in vitro, and use this information to develop new methods for propagating ES cells possibly without the presence of feeder cells. Unpublished data from the applicant’s postdoctoral work (Ying et al., Nature submitted) suggests that self-renewal of murine ES cell is dependent upon LIF signaling in order to counteract MAPK- and GSK3-driven differentiation. Thus LIF signaling is dispensable if the cells are shielded from those differentiation cues. Indeed, it is even possible under those conditions to grow stat3 null murine ES cells. In fact, additional unpublished data from the applicant indicates that inhibition of MAPK can prevent mouse ES cells from differentiating and/or dieing when weaned from feeder cells. This raises the possibility that similar effects may be seen in human ES cells, which would represent a significant technical advance. Overall, the experiments proposed here are highly innovative and potentially highly significant.
STRENGTHS: These are very thoughtful experiments, which are based on significant advances the applicat made as a postdoctoral fellow in understanding the signaling pathways that control self-renewal of murine ES cells. The first series of experiments will explore whether inhibition of MAPK and/or GSK3 can overcome the need of human ES cells for a feeder layer. A second series of experiments is based on the observation that human ES cells are less adherent than murine ES cells, especially in the absence of feeder cells. This presents possible problems for developing feeder independent culture conditions, because less adherent ES cells tend to undergo spontaneous differentiation. Preliminary observations by the applicant indicate decreased integrin expression in human ES cells may underlie this difference. A solid series of experiments will further analyze this difference in integrin expression, and will then test whether ectopic expression of specific integrins affects their adhesive properties, self-renewal, and responsiveness to LIF, bFGF and inhibition of MAPK and GSK3. A third very interesting Aim will explore the requirement of human ES cells for bFGF. Mouse ES cells express the ras family protein Eras, which activates the PI-3 kinase/Akt pathway, and do not require bFGF. Eras null mouse ES cells are bFGF dependent, and human ES cells do not express Eras. The applicant will test whether constitutive expression of activated Eras bypasses the requirement for bFGF by activating the PI-3 kinase pathway and its downstream targets.
The ultimate goal and rationale was well thought out and logical. The project is feasible in the time span and could have major impact on the entire field, at the most basic level.
WEAKNESSES: Although generally well written, the proposal is not well formulated around Aims, and it would have been helpful to more clearly delineate the specific goals for each aim with priorities, pitfall, etc. It should be noted that the exciting preliminary results have not yet been published.
DISCUSSION: This is a new assistant professor at the perfect time for CIRM funding. Three fundamental questions are asked whose answers could potentially have a broad impact. 1) what are requirements for feeder-free hESC? 2) why do human ESC grow poorly on substrates compared to mouse? look at specific integrins. 3) Why do human ESC need FGF? Because they lack RAS? The PI has preliminary data to back up the models and hypotheses that will be tested. One question was about the novelty of this work given that the activation of Wnt pathway drives pluripotency in mES and hES. What is the novelty here if it has already been shown that the STAT pathway is sufficient but not necessary for mES differentiation? Reviewers recommended that the PI look at other grants to learn about the proper format, i.e. organization around Specific Aims would have been helpful.