Recommended if funds allow
Statement of Benefit to California:
SYNOPSIS: This application from a premiere bioengineering lab has some ambitious aims centered on understanding the role of mechanical forces, extracellular matrix, growth factors and glycans in directing stem cell fate. Just this last sentence would be pie-in-the-sky except that the group has developed high throughput techniques for handling large, similarly combinatoric problems, but not in hESC. The specific aims are (i) to test a huge array of microenvironments for endpoints of adhesion, proliferation, maintenance of pluripotency, apoptosis (ii) to develop an array platform for presentation of growth factors and glycans and look at all the endpoints discussed in the first aim and (iii) to analyze shear flow effects on maintenance of pluripotency and endothelial cell differentiation. SIGNIFICANCE AND INNOVATION: Microarray-based, discovery-driven approaches to the study of cell differentiation based on the type of matrix polymers and proteins that cells are adhered to have been shown to be fruitful for a variety of cell types. In this proposal, the investigators will expand this type of microarray technology to also include more soluble factor environments such as growth factors and glycans. This type of study may enable rapid accumulation of knowledge concerning what combinations of factors regulate hESC adhesion, proliferation, maintenance of pluripotency, and differentiation. Furthermore, the investigators will combine the effect of fluid mechanical environments to further regulate cell proliferation, maintenance, and differentiation. The combined effect of chemical and mechanical forces on embryonic stem cell differentiation is understudied and interesting, and there is a potential to identify better conditions for differentiation and maintenance of hESC. STRENGTHS: The PI is an elder statesman of bioengineering and brings his experience with high throughput engineered microenvironments and microfluidics to bear. Significant established infrastructure is available for the work. Other engineering groups are doing similar studies of ECM and other arrayed proteins on hESC fate, but this approach is original. The investigators have significant preliminary results and an excellent track record of research in microarrays, shear stress, growth factors/inhibitors, and glycans. Use of a specific hES cell line that tolerates dissociation strengthens the proposal to perform microarray experiments to screen differentiation conditions. The effect of shear stress on hES cell differentiation is an understudied area of research that is interesting. WEAKNESSES: The work is ambitious for the time-frame, especially given that no single person is devoted full time to pushing the project forward. Since the combinations of conditions examined are so large, and there is some fluctuation in patterning, it would help to have an estimate of the numbers of replicate experiments the PI anticipates. In this respect, the reviewers are not given sufficient information to judge the feasibility of completing the studies in the 2 year time frame. In fact, how the combinatorics of Aims 1, 2, and 3 are handled is not made clear at all, and this makes the proposal difficult to review. Will the glycans in various combinations be combined with the other ECMs and trophic factors in spots on the arrays? Will the shear stress be applied to cells on only a subset of matrices? Just as clinical studies require a power analysis to help reviewers, a similar presentation would have improved the application. In addition, these studies really are screens - whether the research will fundamentally change the way cells are maintained or differentiated will require a hand-off to stem cell biologists. For example, expression of certain markers of differentiation does not mean that the cells generated are fully functional. Nonetheless, even if Aim 1 gets accomplished successfully, and little progress is made on the other aims, the research will yield important information. One concern that was not acknowledged is that different concentrations of the many proteins to be arrayed could have fundamentally different effects. There is no plan for looking at different concentrations, although the applicants have the luxury of using newly derived lines (for example, approved lines generally don’t adhere to 5 ug/ml laminin but do adhere to 500 ug/ml laminin.) The arrays may be presenting different doses of ECM or other proteins, but this is not clearly stated. In addition, the biology collaborator suggests that isolation of certain proteins from Matrigel may be possible for the purposes of the grant, whereas in the body of the grant this possibility is presented as a relative certainty. This may slow the effort more than anticipated. Although the applicants have some preliminary data suggesting that hESC will grow for a week on the glass arrays (though how well is not clear), and they anticipate no technical difficulties, that assumption may be a bit naïve. For example, experienced hESC hands would probably anticipate difficulty getting cells off of a variety of matrices in the same conditions for flow studies - it is likely that the conditions for getting cells to single cell suspension from the arrays may be different depending on what the cells are sitting on. The release of cells is needed for the flow cytometry studies proposed. There are alternative ways to approach this problem, but the problem has not been acknowledged. Similarly, there will inevitably be a learning curve for interfacing these cells to the arrays and choosing the proper incubation conditions, which are not discussed at all even though there have been previous studies of hES cell differentiation on microarrays of different biomaterials (e.g., the Langer lab). Also, the proposed well gasket format of the microarray for regulating the extracellular matrix-growth factor-glycan environment (the soluble factors) does not seem compatible with fluid shearing. DISCUSSION: This is a highly innovative, ambitious proposal from one of best bioengineering labs in the country, which is well known for studying shear stress on cells. They propose to study a huge array of microenvironments, but they already have preliminary data on hESCs. It is highly innovative to bring this work to hESCs. However, the applicant falls into trap of equating the expression of biological markers with having the desired cell type. Other weaknesses include the lack of a plan for handing off the cells after development to a well-established stem cell laboratory, and previous studies in the field on hESC differentiation on microarrays of different biomaterials were not referenced. In addition, while the well gasket format of the microarray was well described, one reviewer did not see how fluid shearing was simultaneously compatible with the arrays of glycan microenvironments. There was a question of whether this work is NIH-fundable, but panel members noted that those older lines can't get into the arrays for shear stress testing, thus the Melton lines may be more suitable for array placement than presidential lines.