The work proposed here will explore stem and progenitor cell populations important for therapeutic angiogenesis and vasculogenesis (new blood vessel growth). This work is especially relevant to cardiac repair following a heart attack, but will contribute to the building a vascular supply for tissue enginneered organs.
Statement of Benefit to California:
The research proposed here could result in new techniques and methodology for the differentiation of endothelial cells optimal for therapeutic vasculogenesis. The citizens of California could benefit from this research in three ways. Some may benefit from the potential for new medical therapies. Others may benefit indirectly from the potential for these technologies to bring new business ventures to the state of California. The third benefit is the education of the students involved in this study, especially in the Central Valley.
SYNOPSIS: The PI has studied the development of vessels from mouse ES cells and this project aims to translate these results to the hESC system. The idea is to compare different “stages” of ESC-derived endothelial cell development to optimize the cell population that would be most proficient at forming new vessels. Four distinct stages are proposed, namely hESCs, Flk1+ progenitors, committed progenitors from newly sprouted outgrowths containing both endothelial and smooth muscle cells, and finally differentiated endothelial cells.
Experiments in Aim 1 will define these stages in the human system. Cells will be purified and characterized by marker analysis. In Aim 2, the functional ability of the cells derived from each stage will be tested for vessel growth both using in vitro collagen gel assays and in vivo using the chick CAM assay. Vessel growth will be quantified. In Aim 3, the PI proposes to evaluate the gene expression patterns for a large list of regulatory genes at various stages of endothelial cell development and then to disrupt the function of some.
INNOVATION AND SIGNIFICANCE: In principle, this project could produce very significant protocols for generating vessels, which could have important significance for using ES cells in therapeutic angiogenesis. Impact could be on repair of ischemic tissue, improving cardiac function, vessel repair, and even for gene product delivery through nascent vessels. It is likely that translation of the results in mouse to the human system will require innovation, but this is not clearly laid out in the proposal.
STRENGTHS: Importantly, the PI has experience in the last few years as a postdoc in generating vessels from mESCs. In addition, the applicant has an engineering background and interest in developing quantitative methods for measuring vessel growth in a 3D context. This is well considered with respect to Aim 2.
The PI is a new investigator who has trained in a top tier tissue engineering lab (Robert Nerem), and this proposal would enhance the transition in a new and promising field. The proposal appears to be largely feasible.
WEAKNESSES: A major issue is whether the preliminary data can be translated directly to the human system and there is concern that the PI has not considered this in enough detail. The basic hypothesis that the endothelial cell progenitor cells are likely to work best, is probably correct. While the first and last groups (ES and EC) may be homogeneous, the middle two are not. Thus, the four “stages” appear to be rather arbitrary. For example, the Flk1+ cell population will be extremely heterogeneous. While the PI indicates that the kinetics of human EC development will differ from the mouse, there is no explanation of how or indication of how this will be addressed. The PI states that the ES cells will maintain their stemness by culture in LIF, which is a further indication that the transition to the human system needs further consideration.
The third aim will evaluate the expression of a large battery of known regulatory genes. Which ones will be deregulated and why? Surely if the expression of Flk1 is blocked the pathway will fail, but what new information will have been learned? The purpose of this Aim is not clear and it is at any rate far too ambitious for the 2 year plan.
DISCUSSION: Reviewers believed there is some level of naivety in the PI - e.g., the proposal to maintain pluripotency of hESCs in LIF. One rveiewer considered the third aim to be very ambitious. The reviewer would like to encourage this applicant but believes the PI is not quite ready for funding. Another reviewer expressed concern that a critical mass effort is not available at the institution and believes that it may be a distraction for the applicant to work in an unknown difficult system at this point. The applicant should be encouraged, however, as bioengineering is a valuable expertise for stem cell research. Some of the weaknesses in the application may be due to not being well-connected and it would be good to get the applicant involved in state-wide stem cell network.