Stem cells can be transformed, or differentiated, into whatever cell the body instructs them to (e.g., bone or cartilage). Biologists have only begun to discover some of the specific chemicals that are used to direct the cells’ development into a given cell type. Our proposal provides a new method for rapidly screening reagents, which will enable us to develope new stem cell differentiating protocols. These new protocols are especially lacking in the area of human embryonic stem cells due to restrictions on federal funds. These protocols can be used by other scientist to enable new studies in other fields of biology and by physicians for regenerative medicinal treatment. This nanotechnology provides a new tool for growing tissue with multiple cell types. If successful, this will enable a new era in tissue engineering where complex functioning tissue can be created from stem cells. These man-made tissues include small blood vesicles for vascular grafts, skin for burn victims, bladder for bladder replacement surgery, and bone-cartaliage interface for treating arthritic patients. Human embryonic stem cells (hESC) show great promise for tissue regeneration because of their propensity to make many of the cell types in the body. However, exactly how hESC can be directed to make specific parts of the body remains unclear. This is a major roadblock to harnessing the striking potential of hESC as a regenerative engineering solution for organ failure. Regeneration of complex tissues derived from the inner layer of the body called the endoderm such as the lung and vascular system is particularly challenging. This work cannot be funded by the Federal Government because we will utilize novel hESC lines. We already have in hand several novel hESC lines that have a strong propensity to enter endodermal tissues in small scale culture systems. We also have already enjoyed preliminary success with other kinds of stem cells in getting them to enter and make endothelial cells in vitro. Now we plan to apply our nanotechnology approach to provide a tissue engineering solution for making small blood vesicles This project will bring together investigators who have extensive complimentary expertise in silicon nanotechnology, human embryonic stem cell biology, organogenesis, pathway analysis, and vascular biology. The experimental techniques described in this proposal lie at the cutting edge of both nanotechnology and human embryonic stem cell biology. They are straightforward extensions of our current capabilities and therefore represent feasible ideas that will produce results quickly. We believe there is unlimited potential for the cross-pollination of the two fields. By leveraging our extensive preliminary results and expertise, we expect to be able to discover a toolbox of critical factors that can direct the growth of hESC into endothelial precursors, which can then lead to the realization of hESC-derived vascular tissue for regenerative medicine.
Statement of Benefit to California:
Cardiovascular disease, including coronary artery and peripherical vascular disease (small and medium caliber arteries), is the leading cause of mortality in the United States each year and over 500,000 coronary artery bypass grafts are performed annually. Therapies for coronary artery and peripherical vascular diseases often require replacement of the diseased vessels with vascular grafts. Autologous arteries or veins are the best substitutes for small diameter (<6 mm) vessels. However, many patients do not have vessels suitable for grafting due to preexisting vascular diseases or vessel use in previous procedures. Therefore, many attempts have been made at engineering a small caliber arterial substitute that include endothelial cells (EC), smooth muscle cells (SMC), and/or fibroblasts on a variety of tubular scaffolds with limited success. This proposal seeks to discover a toolbox of critical factors that will enable hESC to enter, integrate in and function as part of tissue regenerative solutions for vascular grafting. If we are successful, these critical factors may become medicines that will be critical to harnessing the potential of hESC as tissue regeneration solutions. The known-how and intellectual property generated by this research will reside in California, thus giving the state a competitive advantage over other states in area of stem cell research. The cumulative effect of scientific and newspaper publications and conference presentations will establish California as a focal point for future investments in this important emerging field.
SYNOPSIS: The biological focus of this application is to develop strategies that differentiate human embryonic stem cells into vascular smooth muscle and endothelial cells. The long-term goal is to form vascular tubular structures ex-vivo. The proposal is built around nanotechnology in which nano-cavities are etched in a silicon microchip and then sealed with a lipid bilayer. Through spatial and temporal regulation of the release of reagents from these nano-containers, the investigators aim to control hESC differentiation into endothelial cells and smooth muscle cells with single cell resolution. The proposal has 3 specific aims: 1) to calibrate and optimize the ability of the lipid bilayer to release chemicals from the nano-cavity under applied voltages using fluorescence microscopy; 2) to establish a purely chemical differentiation protocol for differentiating individual embryonic stem cells toward the endothelial and vascular smooth cell lineages leaving neighboring stem cells unchanged; and 3) to grow tissue with vascular smooth muscle on one side and endothelium on the other to produce an artificial vascular wall by filling different regions of the nano-cavity array with various differentiating reagents. SIGNIFICANCE AND INNOVATION: This proposal will develop an array of nanoscale wells filled with growth factors and other reagents that regulate hES cell differentiation and covered by a lipid membrane. Each of these small containers can be opened by application of small electrical fields that burst the thin lipid membranes. This is an innovative proposal in which the investigator proposes using state-of-the-art technology to explore the differentiation of human embryonic stem cells. The main innovation stated is the development and application of these lipid bilayer electro-fluidic valves to expose select cells to differentiation agents. Its significance is reflected in the fact that vascular disease indeed is a major medical problem. STRENGTHS: Dr. Cronin is a well-trained young investigator who, based on his peer-reviewed publications, has made significant contributions in the field of nanotechnology. He has identified a collaborator, David Warburton, who is a senior, highly established developmental biologist. The described nanotechnology once developed will be an interesting tool for performing basic studies on the effect of exposing distinct cells to distinct reagents to see how the treated cell and surrounding cells differentiate. The technology gives unprecedented spatial and temporal resolution in how cells are exposed to different chemicals. Methods for evaluating cellular responses and differentiation are specific and well-described. WEAKNESSES: Despite focusing on development of microchip technology for screening “hundreds of chemicals” for their differentiation capacity, there is little discussion in the proposal as to which chemicals will be screened. Furthermore, there is no indication as to the biological basis for the choice of a particular reagent. There is a brief description of stem cell differentiation reagents, which includes the standard cytokines and growth factors, but there is no indication as to how the investigation will be developed to identify new chemicals or explore combinations of reagents. In addition, the formation of the containers seems well established, but there is little description of how different reagents will be filled into different adjacent wells, especially hundreds of different reagents into hundreds of different wells. The type of scientific hypothesis that will be tested with the described technology is also vague. How will the unique capabilities be taken advantage of? Cells can be exposed to different combinations of reagents using a variety of other methods. The significance of having such high resolution of point sources of such small volumes of reagents seems even weaker for the stated future translational research, especially if the goal is to pattern differentiation of cells in different regions towards construction of small diameter blood vessels, which are formed over relatively large (millimeter-scale diameter and centimeters long) areas. Why is single cell resolution necessary? The stability of the lipid bilayer barrier over the timeframe that may be necessary (up to weeks) to control differentiation of hES cells is also worrisome. Finally, if formation of the required cell types is successful, how will the resulting tissue be subsequently removed from the substrate? DISCUSSION: The goal of the proposal is to develop differentiation strategies for vascular tissue. The approach aims to utilize nanotechnologies etched in lipid bilayer chips. Vascular tissue would be juxtaposed next to endothelial wall. This is innovative technology and has some merit, but the applicant did not adequately focus on the biology and appropriate details were lacking. For example, there was no discussion of what chemical libraries would be screened, and no discussion of what the collaborator Dr. Warburton would do. In addition, neither endothelial cell development nor how the matrix would be constructed are being studied in this proposal. The technology is not a good match for the desired outcome because it does not have a biological focus. The applicant has not published in the particular area of nanotechnology that he proposes to apply, and there is no indication that the developmental biologist collaborator contributed to thinking about the biology in the proposal. Reviewers noted that the applicant could end with an adjacency of cell types through the proposed approach, but it is unclear how they would end up with vascular structure.