Technology platform to develop defined culture conditions for the derivation and expansion of hESC lines
Tools and Technologies I
Advances in research on human embryonic stem cells (hESCs) will have major impacts on the quality of life of millions of people with health problems such as cancer, cardiovascular disease, and neurodegenerative disorders (e.g. Alzheimer’s and Parkinson’s diseases). The California Institute of Regenerative Medicine was established to develop novel cell-based therapies to treat these and other presently incurable disorders. With their ability to develop into virtually all adult cell types, hESCs represent the “raw material” for many cell-based therapies. To realize the full potential of hESCs in regenerative medicine requires, among other things, (1) establishment of well-defined culture conditions for their growth and differentiation, (2) cost-effective protocols for their expansion, and (3) derivation of new pluripotent stem cell lines under completely defined conditions. In this grant application we propose a series of experiments and the development of a novel technology platform that will provide critical information and protocols for hESC researchers. Previous studies on factors affecting stem cell growth have focused on only one or a few elements of the cellular microenvironment, e.g., single extracellular matrix components or growth factors. Using such approaches, the screening of large numbers of factors in many combinations would be cost-prohibitive. Furthermore, currently existing hESC lines are not suitable for therapeutic use because they have been grown in poorly defined conditions containing animal-derived products, which may harbor pathogens. The proposed research will develop a high-throughput cellular microarray screening tool that incorporates combinations of recombinant proteins, synthetic materials and nanopatterned surfaces. This tool will allow the cost-effective, concomitant screening of the effect of thousands of conditions on growth and maintenance of hESCs. We combine biochemical and physical microenvironments in a controlled manner to study the responses of hESCs to physicochemical modulations of their signaling behavior and cellular fate. The results from our studies will provide fully defined and optimized culture conditions for the derivation and expansion of new hESC lines without exposure to animal-derived products. In summary, we will develop a comprehensive approach to elucidate the responses of hESCs to a variety of factors in the microenvironment. Application of this novel and powerful technology will lead to the definition of the optimal parameters for the control of hESC growth. In addition, this technology will facilitate research on the directed differentiation of hESC into specific mature cell types, such as neurons, cardiomyocytes, pancreatic islets, and other cells, that can be applied in the treatment of a variety of debilitating human diseases.
Statement of Benefit to California:
The rise in life expectancy of the U.S. population to over 80 years will likely lead to an increase in the number of people suffering from degenerative diseases. Current medical treatments can control, but not cure, disease such as cancer, heart diseases, Alzheimer’s, and Parkinson’s. Recent advances in the study of pluripotent stem cells have provided the opportunity to develop novel strategies involving cell replacement therapies for the treatment of many currently incurable diseases and may overcome the inadequacy of the conventional drug-based treatments. Developing cell replacement therapies requires sufficiently large numbers of clinical grade human embryonic stem cells (hESCs) that can be thoroughly tested and characterized. To address this critical need we have developed a comprehensive and cost-effective technology for the high-throughput screening of conditions that regulate hESC proliferation and differentiation. The aim of our current research is to extend the capabilities of this technology platform and apply it to systematic investigation of the physicochemical conditions controlling hESCs proliferation. Our technology will allow the screening of thousands of well-defined parameters to select the optimal culture conditions for stem cell proliferation and also differentiation. Our systematic study on these parameters, including recombinant proteins, synthetic molecules and nanopatterned surfaces, will enable the development of cost-effective large-scale production of hESCs. In addition, our study on synthetic biopolymers and peptides will eliminate the need for animal-derived products that are currently used at early stages of hESC derivation and pose potential problems for human therapies. Our technology, which we will make freely available, will additionally benefit many other lines of scientific inquiry, such as defining growth conditions of rare adult stem cell populations and modeling the cellular basis of diseases. Thus, our proposed research is fundamental to applications of hESCs in regenerative medicine and has broad benefits to researchers with a wide spectrum of scientific interests. Our research will not only benefit the health of Californians, but also the California economy by enhancing and generating local businesses. In addition, the outcome of this project will lead to the development of a biotechnology platform that can provide great benefits to the advancement of California biotechnology. The patents, royalties and licensing fees that result from the advances in the proposed research will provide California tax revenues. Thus, the current proposed research provides not only the essential foundation for the scientific advances in regenerative medicine to improve health and quality of life, but also potential technology advancement and financial profit for the people in California.
This goal of this proposal is to screen and identify key factors in the cellular microenvironment that will enable improved growth and maintenance of undifferentiated human embryonic stem cells (hESC), and to use this information to derive new hESC lines. In the first aim, multiple combinations of growth factors, surface molecules and extracellular matrix components will be printed onto a solid support, and each condition will be evaluated for its ability to promote growth of human stem cells and maintenance of pluripotency. To complement this approach, a second aim will employ a similar strategy using arrays of synthetic polymers and scaffolds to assess the effects of charge, hydrophobicity and topography on stem cell growth and maintenance. The third aim proposes to derive a novel, self-renewing, hESC line using information gleaned from aims 1 and 2. The applicants comprise an academic, multidisciplinary team with expertise in bioengineering, nanotechnology and reproductive medicine. Current techniques for growing, scaling up, and maintaining undifferentiated hESC cultures are very limited, and technologies that could potentially overcome this roadblock are extremely desirable. This proposal offers a straightforward, innovative means for addressing some of these hurdles, and if successful, would be of high impact. Reviewers were most enthusiastic about the first aim, as printed arrays offer a potentially improved format for testing combinatorial variations of growth components, something that is difficult to achieve with typical solution-based methods. The use of arrays to identify optimal conditions for maintaining hESC cultures over time, as judged by detection of known pluripotency markers and teratoma formation, represents a simple yet elegant strategy for dissecting the chemical, biological and structural components that underlie these properties. Based on preliminary data, the reviewers were confident that the goals of this proposal are readily achievable, although the full two-year time frame might be necessary to realize Aim 1 alone. While intrigued by the technological platform, some reviewers felt that Aim 2 represented a significant detraction from the overall proposal. One reviewer felt that the added complexity of a topography screen might be unwarranted, as the stated goal of Aim 1 is a protocol for long-term self-renewal in culture, a sufficient goal in and of itself. Furthermore, the rationale for choosing the particular topographies to be explored was not adequately justified, and reviewers were uncertain as to how and why these arrangements were chosen. Overall, reviewers felt Aim 2 might have been stronger if the parameters to be tested were a) larger and less biased, or b) smaller and more biologically motivated. The principal investigator has a remarkable track record in the areas of bioengineering, extracellular matrices and cell biology and has assembled a strong team of experts in reproductive medicine and nanotechnology. The reviewers expressed great confidence in the ability of this team to meet the challenges presented in this application. Overall, this is a straightforward, feasible proposal that was clearly presented by a principal investigator with a proven track record of success. The specific aims were judged to be disproportionately meritorious, but the potential for high impact findings bolstered the reviewers’ enthusiasm. PROGRAMMATIC REVIEW A motion was made to move this proposal to Tier 1, Recommended for Funding. Despite the weakness of the second aim, reviewers felt that the strength and simplicity of the first aim, combined with proven track record of the investigative team, warranted additional consideration. Reviewers discussed whether identifying ideal conditions for adherent growth, i.e. molecular matrix formulations, could address the scale-up deficiencies that currently define the field, or whether it would be preferable to focus efforts on improving solution-based techniques for use in bioreactors. Some felt that it would indeed be desirable to have a matrix array for scaling up growth, and the use of bioreactors does not necessarily preclude the use of an adherent culture system, such as one where cells are attached to the surface of a bead within a bioreactor. Based on the strength of the team and encouraging preliminary data, as well as the potential for addressing a current roadblock in cell growth and maintenance, the motion carried.