Funding opportunities

A Novel Engineered Niche to Explore the Vasculogenic Potential of Embryonic Stem Cells

Funding Type: 
New Faculty I
Grant Number: 
Principle Investigator: 
Funds requested: 
$2 108 683
Funding Recommendations: 
Grant approved: 
Public Abstract: 
Statement of Benefit to California: 
Review Summary: 
SYNOPSIS: This proposal aims to explore enhancements of vessel growth into ischemic tissues, such as myocardial infarct, by delivery of an appropriate cell type to stimulate formation of host blood vessels. The aims of this proposal build on previous and ongoing work performed by the PI using novel engineered 3D structures (‘instructive biomaterials’) to promote angiogenesis from mesenchymal stem cell (MSC) populations. The PI’s long-term goal is to develop instructive biomaterials and strategies to direct tissue repair. The grant application is extremely well-written. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This is a highly significant and innovative project proposed by a productive young investigator. Regenerative medicine will only reach its full potential when novel methods of cell recruitment and direction are developed. The current static methods of cell culture will not get us there. This project addresses these shortcomings head on by developing three-dimensional milieus for study. The problem to be studied – restoration of vascularization – has relevance to most tissues, native and engineered. Further significance comes from the expected increased understanding of the effects of extracellular matrix on hESC differentiation. Finally, investigation of the role of hESCs in vasculogenesis – direct or supporting – is significant for therapeutic use of these cells. Innovation is mostly in the attempt to engineer a niche suitable to study vascular differentiation of hESCs. A very significant strength of this proposal is that the applicant has strong engineering background and skills and also understands the biologic problem and approaches with the sophistication of a biologist. The PI proposes to develop an in vitro system (a microfluidic “niche”) to study the endothelial differentiation of hESCs, and to study purified populations of the resulting endothelial cells in a rodent model of hindlimb ischemia. Aim 1 combines the 3D structures developed by the PI (and similar structures are now being used in many bioengineering labs) with a microfluidics device developed by the collaborator, in order to be able to deliver morphogen gradient signals to differentiating stem cells. Gradient morphogen signaling is important in stem cell differentiation but not capturable in current tissue culture paradigms. Potential problems and solutions in this aim are well described. In terms of novelty, several other groups have reported engineered delivery of VEGF and bFGF and enhancement of angiogenesis, migration, signaling, etc in various stem cell populations, though not in human ES cells to date. One unacknowledged technical issue is the relative unfriendliness of PDMS (even if coated with fibronectin and collagen and the surface functionalized with the air-plasma treatment) to human ES cells compared to other stem cells. In a lot of PDMS applications (despite publications from some labs), matrigel is still required especially if no feeder layer is used. Aim 2 is described as the confirmation of two competing hypotheses but really the two hypotheses are not incompatible. Some growth factors can work in autocrine and paracrine fashion (including bFGF), and VEGF and PDGF may also have both autocrine and paracrine activity depending on the expression of the right cellular machinery. The necessity of an intermediate embryoid body (EB) step implies a lot of noise in the differentiation protocol because of the varying sizes of the EBs generated. Since the PI is using a microfluidic device, he could control the size of the EBs and take noise out of this step (as reported by the Takayama group last year). In Aim 3 the PI acknowledged that no one in his lab has experience with the hindlimb ischemia model and that could present an issue for this aim. The writing of the detailed experiments is telling regarding the inexperience in that the PI does not state where the cells will be transplanted in the animals, nor does he state the anatomic structure that will be analyzed in the histopathology experiments. What are the controls for these studies? In an important piece of preliminary data, Figure 5, showing integration of human engineered capillaries into the mouse (mouse red cells are carried), there is absolutely no indication of the efficiency of the process. Given that a major strength of this grant is the quantitative tools brought to the table, some estimate of efficiency would help with feasibility of the tools leading to translation. Overall, the research plan is well developed, logical and described in great detail. The investigators demonstrate excellent understanding of endothelial differentiation of hESCs and of controlled in vitro studies of cell interactions with biomaterial substrates. The parameters and their ranges are specified and the rationale is clearly stated. The analytical methods are appropriate and comprehensive. Some minor problems are in the understanding how exactly the cells will be seeded and cultured in the microfluidic device, and how the co-cultures will be established and monitored. The anticipated difficulties and backup strategies are clearly described. Preliminary studies are extensive and adequate to establish the feasibility of the proposed approach. This is an excellent proposal with strengths that include high significance, well developed research design, strong preliminary studies, strong investigators. The studies, and potential problems, are well described and the assembled team has a high likelihood of completing the studies. Some weaknesses include somewhat unclear details in the design of the engineered “niche”, and some uncertainty that the highly purified cells can in fact be derived. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Putnam is Assistant Professor of Biomedical Engineering and Chemical Engineering and Materials Science. He holds three degrees in chemical engineering, followed by a post-doc year in cell biology. He is an ideally suited young investigator to develop the novel therapeutic approaches essential to regenerative medicine. Dr. Putnam's career goals are carefully laid out and include a self-organized mentoring committee that will listen to progress on this project twice a year. He is excited by his unique position at the interface of engineering and biology and plans to make major progress in the field of regenerative medicine. The PI has an excellent publication record in the area of research, and is already well funded for a junior investigator. The PI has an RO1 investigating MSC angiogenesis in engineered 3-D fibrin matrices, and the role of MMPs in capillary formation. He also has an NSF career award examining MSC angiogenesis and the role of mechanical stimulation in angiogenesis in an engineered 3-D hydrogel. A second RO1 on tumor angiogenesis in engineered structures is pending. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant has put together a group of mentors which is a great idea for any assistant professor, and the group is a major factor in his future success. He presents a detailed plan for how he would like his lab to grow and what his stature should be over the next few years. His local and broader campus contacts and the UCI environment are extremely well-suited to fostering the work. The institution has made huge effort to be a leader in stem cell biology, and they are fast becoming a powerhouse in this area. Dr. Putnam's application includes several enthusiastic letters in support of his project and his faculty position at UC Irvine. The institutional commitment is strong. DISCUSSION: Reviewers agreed that this is an excellent, well-written application by a very successful candidate with a good career plan. The PI has a strong engineering background combined with very good understanding of biology. The preliminary studies are strong and provide great confidence that the research will be productive. The PI has collected a good team of mentors. A couple of minor criticisms in the research proposal were cited such as not having incorporated all available technologies (e.g., could use microfluidics to control the size of the EBs) and the lack of direct experience in hind leg ischemia animal model. Overall, this is a strong proposal and one reviewer felt it was a perfect applicant for this RFA.

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