Establishment of novel defined culture conditions for human embryonic stem cells and induced pluripotent stem cells
Tools and Technologies I
Human embryonic stem (hES) cells can give rise to virtually all types of specialized adult cells in human body. Thus they are thought to be the potential source of the cell-transplantation therapy for the treatment of diseases such as Parkinson's disease, myocardial infarction, and diabetes mellitus. While hES cell research has made a considerable progress in developing methods to generate specialized differentiated cell types including neurons, cardiomyocytes, and insulin-secreting cells, studies to identify the key mechanisms that allow hES cells to maintain such an enormous differentiation capacity (pluripotency) have just begun. Because current culture methods to amplify undifferentiated hES cells largely rely on animal-derived materials, it is critical to eliminate such a potential pathogens from hES cell culture environments in order to use hES cells for cell therapeutic purposes. We have recently found that a specific signaling pathway (like a hormone that mediates various biological information) controls cell-cell and cell-matrix attachment of hES cells. The goal of the proposed study is to develop novel technologies by which hES cells can be propagated under completely defined conditions by modulating the specific signaling pathway. We will also apply this technique for propagation and generation of induced pluripotent stem (iPS) cells that can be produced from adult differentiated cells. These studies will contribute to establishing key platforms for translating pluripotent stem cell research into medical applications.
Statement of Benefit to California:
Our research will focus on developing novel technologies by which hES and iPS cells can be propagated under completely defined conditions. The establishment of such a new method would impact virtually all hES and iPS cell-based application programs as it involves a common basic process to expand undifferentiated pluripotent stem cells before turning into any type of adult cells for the therapeutic purposes. It is therefore predictable that the new methodology will be promptly translated as an intellectual property to be commercialized, and would substantially activate the biotechnology field in the State of California. More importantly, the new methodology will be provided to the Institutes in California at the highest priority where the method will accelerate the process to apply the pluripotent cell-based transplantation approach for the clinical settings that would further substantiate the enhancement of the medical environment for California citizens.
This application is focused on the development of cell culture methods to cultivate both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) in defined, feeder-free conditions. The goal is to establish highly scalable conditions for growing stem cells without animal-derived products. The proposal builds on the Principal Investigator’s recently published work showing that hESC previously grown on feeder cells can maintain pluripotency for extended periods on a synthetic matrix in a chemically defined medium with the addition of a commercially available inhibitor. These cells, while pluripotent, have a reduced growth rate compared to those grown in traditional media. The PI proposes to develop improved culture conditions to support higher growth rates by testing alternative synthetic matrices and inhibitors. These improved conditions would then be tested on multiple hESC lines to ensure general applicability. In the second aim of the proposal, these experiments are extended to human iPSCs. The applicant proposes to first test gain- and loss-of-function in the signaling pathway on the pluripotency of these cells. Then the applicant would test the culture conditions derived from Aim 1 on iPSCs obtained from other labs, and finally, attempt to derive iPSC lines de novo using these conditions. The reviewers raised doubts about the novelty and impact of this proposal. They noted that many other groups are working to develop improved culture conditions for human pluripotent cells and weren’t convinced of the advantages of this particular approach. In addition, they thought that the work was feasible but raised some concerns about the research team and budget. Overall, they felt that the project does not sufficiently advance stem cell technology. The reviewers agreed that the application focuses on an important roadblock to creating clinically relevant hESCs and iPSCs: scalable generation, growth and expansion in an animal product-free environment. But while the goal of this proposal is an important one, the reviewers questioned the impact of its specific approach. One reviewer questioned the time and effort proposed in Aim 1 for a marginal potential improvements in growth rate compare to the current method. Another reviewer criticized the small number of hESC lines that would be used to develop a “universal” culture system: just six cells lines, five of them derived by two research groups. Testing a larger number of cell lines from multiple labs and countries would be more informative. The reviewers agreed that the proposal is feasible and that the recently published data is compelling. One reviewer commented that the overall methodology and experimental design is well presented in the application, but other reviewers raised questions. One was disappointed that the rationale for choosing specific inhibitors or for the choice of substrates was not thoroughly described. The reviewer suggests the inclusion of smaller, synthetic substrates in the screen as well as other defined growth media. The PI is a relatively new investigator with excellent training and a good publication record. The PI mentions collaboration with an expert in the field of stem cell biology but there is no letter of collaboration provided or details of how collaboration would be managed. Reviewers found the budget generally appropriate but commented on the high percentage of consumables and excessive staff given the scope of the project. Overall, while this proposal addresses an important roadblock to moving stem cells into the clinic, the particular approach lacks sufficient novelty and impact.