Cardiovascular diseases account for an estimated $330 billion in health care costs each year, afflict 61.8 million Americans, and will account for more than 1.5 million deaths in the U.S. this year alone. A number of these diseases are characterized by either insufficient blood vessel growth or damage to the existing vessels, resulting in inadequate nutrient and oxygen delivery to the tissues. The most common clinical example of this is a heart attack, or myocardial infarction, typically caused by blockage of a coronary artery. The resulting ischemia (reduced blood flow) induces irreversible damage to the heart, leaving behind a non-functional scar tissue. Efforts to restore blood flow to ischemic tissues have largely focused on the delivery of protein growth factors (called pro-angiogenic molecules) that stimulate new capillary growth. An alternative approach is to deliver an appropriate cell type that can either accelerate the recruitment of host vessels or can differentiate into a functional vasculature directly. While adult stem cells have shown promising potential with respect to the former, the potential of embryonic stem cells (ESCs) with respect to either of these two possibilities remains unclear. Therefore, this proposal seeks to: 1.) Utilize a novel, highly tunable, 3D engineered niche to investigate how changes in multiple instructive signals coordinately govern the differentiation of ESCs into capillary vessels; 2.) Exploit knowledge gained from basic studies using this model system to generate a purified population of ESC-derived endothelial progenitor cells (EPCs) and test their potential to repair ischemia in vivo. Specifically, in Aim 1, we propose to further develop and characterize our artificial engineered niche for fundamental studies on ESC fate decisions. Aim 2 will use this system to test two competing hypotheses, namely that: 1.) ESCs can facilitate capillary morphogenesis in an indirect manner, in much the same way as adult stem cells; or 2.) ESCs can be directed down an endothelial-specific lineage by manipulating one or more instructive signals. Finally, Aim 3 will utilize our engineered niche to generate a purified population of ESC-derived EPCs and then test their ability to enhance perfusion in an animal model. Successful completion of these proposed aims may transform the clinical use of stem cells for cardiovascular disease and other ischemic pathologies by enabling identification of those factors and conditions which promote vessel formation. The versatile artificial engineered niche developed here will also yield a new tool that could enormously benefit efforts to screen the combinatorial effects of promising therapeutic compounds. Completion of the planned studies will greatly facilitate the PI’s long-term goal of developing “instructive” biomaterials and strategies to direct tissue repair.
Human embryonic stem cells (hESCs) are pluripotent stem cells that can theoretically give rise to every cell type in the human body. Their potential use for the treatment of human diseases has been heralded with great fanfare and even some controversy. However, their therapeutic potential has yet to be realized due to an incomplete fundamental understanding of the factors that govern their differentiation. This proposal describes studies intended to assess the ability of hESCs to develop into blood vessels; in particular, capillary networks that are responsible for the delivery of oxygen and essential nutrients to all tissues in the human body. This focus is motivated by the fact that cardiovascular disease accounts for an estimated $330 billion in health care costs each year, afflicts 61.8 million Americans, and will account for more than 1.5 million deaths in the United States this year alone. It is the number one killer in this country and in California. Since many cardiovascular diseases are characterized by either insufficient blood vessel growth or damage to the existing vessels, a therapy based on hESCs could have enormous benefit to the citizens of California, the United States, and the rest of the world. Therefore, this proposal has two primary goals. First, we seek to develop a novel technology to systematically investigate the influence of multiple instructive signals on the ability of hESCs to differentiate into capillary vessels. Second, we propose to exploit knowledge gained from the basic studies using this technology to generate a purified population of hESCs and test their potential to repair ischemia (lack of blood flow) in an animal model. Successfully achieving these goals will benefit the citizens of California in three significant ways. First, our efforts may help to transform the clinical use of stem cells, not only for cardiovascular disease but other diseases as well, by enabling identification of those factors and conditions which promote hESC differentiation. Second, the versatile technology developed here will yield a powerful new tool that could enormously benefit California’s biotechnology companies in their efforts to screen the combinatorial effects of promising therapeutic compounds. Third, we expect the proposed studies to directly benefit 8-10 researchers in training and indirectly trickle down to hundreds of undergraduate students [REDACTED] enrolled in courses taught by the PI. This final benefit may perhaps have the most significant long-term economic impact by training and inspiring future leaders to pursue research and development positions in California.
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.