Recommended if funds allow
Statement of Benefit to California:
The underlying hypothesis of the work is that mechanical matrix force is a potent regulator of stem cell fate, specifically through small RhoGTPase Rac signaling. In this proposal the specific aims are to (1) Examine the growth and differentiation of HES into mesoderm, endoderm, ectoderm as a function of matrix compliance (stiffening) of the laminin substrate in two dimensions (2D); (2) Examine the role of matrix compliance in 3D on the RhoGTPase Rac signaling pathway using laminin-conjugated PEG gels; and (3) Build a bioreactor which can be used to precisely regulate loading and mechanical forces on contained HES cultures, to determine how these forces regulate cell fate. SIGNIFICANCE AND INNOVATION: The importance of ECM on hESC renewal and differentiation is well known. The importance of the 3D environment in some aspects of hESC culture (e.g. in EB) is also established. There is also no doubt, both microscopic (at cellular level) and macroscopic (at scale much greater than single cell) mechanical force will affect hESC behavior. However, the evidence is scant that macroscopic mechanical force (the subject of the proposed study) plays an important role in hESC proliferation, renewal or in lineage commitment aspect of differentiation. The innovation is high since the group has technical skills that are unique for building the bioreactor, and analyzing the outcome based on precisely defined imposed mechanical forces. The bioreactor will apparently be based on one constructed in the past by one of the co-investigators. STRENGTHS: -Strong expertise in biomechanical force and in employing 3D system as a model ECM -Aided by expert in hECS to the PI’s expertise -The results of investigation will definitely answer the question whether mechanical forces (in the physiological range) affect hESC behavior or not. The strength of the proposal is the team that has been brought together. Since the most important part of the proposal for the HES cell field is the construction of the bioreactor, that work should start earlier than outlined in the experimental timeline. -The bioreactor may find other applications. WEAKNESSES: The list of assays to be done is huge (and without paragraphs nearly impossible to read) and the grant needs a bit more focus. The idea of defining basic lineage determination and focusing on one or a few signaling pathways should help to guide the research. For the endodermal lineage identification the proposal is to stain for AFP and alpha-fetoprotein (?). It would have helped the proposal if the PI could state why the many bioreactors built to date are insufficient for the studies proposed. The dependence on novel biomaterials can have many unexpected downsides. Tissue engineering has been stymied by the unpredictable effects of materials used in easily manufactured environments. Nonetheless as a leader in this area, the choice of materials will be dictated by the local experience as well as that in the literature. • No prior experience in hESC. • PA gel, even conjugated with laminin, may not be appropriate for hECS. However, that’s the premise of the proposal. • The selection of force parameter range appears to be arbitrary. • The 3D culture method is sketchy. Will hESC survive the preparation condition? • Although the dynamic compression is interesting, the conditions to be tested are not necessarily physiologically relevant. DISCUSSION: One discussant pointed out that there was real mathematical analyses to be employed and that the investigator had picked the right system to use to apply such mathematical analysis. It was noted that the preliminary data from the lab is compelling and that force is likely mediated through RhoGtPase Rac, given compelling data from other labs. Also noted that laminin in 2d, 3D matrices is extremely morphogenic. In sum, this proposal represents mathematical strength, coupled with good matrix cell biology with proven effect on hESCs.