The goal of this research is to develop a new generation of small-diameter vascular grafts to contend with the narrowing of blood vessels that leads to various ischemic complications particularly in the central and peripheral nervous system and the heart. These grafts will be based on stem cell and biomimetic nano-matrix technologies. The investigators note that in the US alone, there are more than 400,000 coronary and 100,000 lower extremity bypass surgeries performed annually. Regrettably autologous grafts are not always available for these procedures, and the failure rate of the surgeries is unacceptably high. The applicant will develop grafts using mesenchymal stem cells (MSCs) and will also explore the potential of human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived endothelial cells as cell sources for vascular graft construction. The applicant will address the following critical issues: (1) need for a scalable and well-characterized MSC source and other potential cell sources (hESC, iPSC) for vascular grafts; (2) optimal nanostructure of the scaffolds for vascular grafts; (3) anti-thrombogenic properties of MSC-seeded grafts; (4) immune-acceptance of the grafts; (5) endothelialization on the luminal surface of the grafts; (6) long-term patency and remodeling of the grafts; and (7) safety issues (the fate of transplanted MSCs or ESCs). The research team will first use a rodent model to optimize the vascular grafts and determine the patency, remodeling, immune-acceptance, and MSC fate of the grafts, and then proceed to a more clinically relevant model that is closer to human physiology (and pathology). To thoroughly understand the therapeutic efficacy of autologous, allogeneic and xenogeneic MSCs in vascular grafts, the three cell types, MSCs, hESC-derived and iPS-derived cells, will be compared in a clinically relevant model. The proposed research will be done within the context of three specific aims: first swine and human MSCs will be characterized in vitro, and procedures for production of nanofibrous scaffolds and cell seeding protocols will be optimized. Then, the performance of MSC-seeded vascular grafts will be investigated in vivo using a rat model and a more clinically relevant model (in Specific Aims 2-3). The rat model (without immunosuppression) will be used to: (1) compare with results from athymic rats; and (2) screen MSC and graft prototypes before proceeding to the more advanced model studies. Major issues including long-term patency, graft remodeling, anti-thrombogenic properties, immune responses, and MSC fate in vivo will be addressed. In addition, the research team will explore the relative potentials of hESC- and iPS cell-derived ECs as a cell source for vascular graft construction in Specific Aim 4, and then compare the results with those from MSCs. Reviewers thought the overall rationale, medical need, and overall experimental design were sound, and felt that the potential impact was high. The preliminary MSC data were considered promising; however, in general, reviewers felt that the proposal did not significantly benefit form the iPS and hESC work, and the rationale for work on the pluripotent cells was not convincing. Reviewers viewed the bioengineering experience of the group as a primary strength of the application. Nonetheless, reviewers expressed serious concerns that the proposed project lacked novelty, and they were unconvinced that the proposed approaches to vascular engineering offered advantages or innovations over other approaches in a very active area of translational research. Reviewers felt that the work was not at the cutting edge of the field. Several technical concerns over the methods of analysis of mechanical function of the grafts also lowered enthusiasm for the proposal. The research plan was considered to be well-designed overall such that the time lines and milestones were achievable. Some reviewers felt that potential pitfalls and alternate strategies were not adequately addressed. One reviewer pointed out that a goal of the research was a product that could be used “off-the-shelf” but that the design of constructs that could be stored “on the shelf” was not clearly considered or described. Further, although the goal diameter of vascular structures was meant for small diameter lumen vasculature, the specific application discussed was a vascular graft for dialysis access, and these are large lumen structures. Methods to rigorously assess mechanical function and responses of engineered cardiovascular tissues and performance parameters (like burst pressure) were not adequately addressed in the research plan. This lack of sufficient information on mechanical analysis was considered a significant flaw of the proposal. Furthermore, the design criteria for the mechanical properties of the engineered vessels were not explicitly stated, and no specific information was provided about quantitative metrics of analyses. Since preliminary data regarding mechanical analyses were not sufficient to convince reviewers of likely success in Aim 1, suboptimal mechanical function of the grafts in Aim 1 could prevent progress in Aims 2 and 3. Aim 4 was underdeveloped and out of place in the context of the rest of the proposal. The PI noted that degradation may take years and stated that weight loss will be analyzed; if this is the case, the stated objectives will not really be completed within the 3 year time frame of the proposal. The PI and key members of the research team have the training and experience to conduct the proposed research. The PI is an Associate Professor of Bioengineering and will commit 25% effort to directing the project and coordinating collaborations. The PI has organized a multi-disciplinary team including expertise in transplant immunology, although the immunology expert has committed only 5% effort to the work. A full time post-doc for the project has significant stem cell and biomaterials experience pertinent to the proposed research. Other personnel have expertise in MSC, and engineering of scaffolds, surgery, and mechanical characterization of the biomaterials and grafts. Collectively, time and effort are generally sufficient and budget is adequate, though reviewers were concerned that in some cases, the roles of investigators were not clearly defined. Furthermore, budget detail was often insufficient. Collaborations, resources and environment were judged as appropriate. The PI specifically stated that the proposed research has no overlap with a currently funded NIH R01 grant, yet several aspects of the proposed work appear to be similar or directly overlapping with the stated aims of the current NIH award. A greater distinction between the two studies should have been clearly articulated.