$16 000 000
Induced pluripotent stem cells (iPSCs) have the potential to differentiate to nearly any cells of the body, thereby providing a new paradigm for studying normal and aberrant biological networks in nearly all stages of development. Donor-specific iPSCs and differentiated cells made from them can be used for basic and applied research, for developing better disease models, and for regenerative medicine involving novel cell therapies and tissue engineering platforms. When iPSCs are derived from a disease-carrying donor; the iPSC-derived differentiated cells may show the same disease phenotype as the donor, producing a very valuable cell type as a disease model. To facilitate wider access to large numbers of iPSCs in order to develop cures for polygenic diseases, we will use a an episomal reprogramming system to produce 3 well-characterized iPSC lines from each of 3,000 selected donors. These donors may express traits related to Alzheimer’s disease, autism spectrum disorders, autoimmune diseases, cardiovascular diseases, cerebral palsy, diabetes, or respiratory diseases. The footprint-free iPSCs will be derived from donor peripheral blood or skin biopsies. iPSCs made by this method have been thoroughly tested, routinely grown at large scale, and differentiated to produce cardiomyocytes, neurons, hepatocytes, and endothelial cells. The 9,000 iPSC lines developed in this proposal will be made widely available to stem cell researchers studying these often intractable diseases.
Statement of Benefit to California:
Induced pluripotent stem cells (iPSCs) offer great promise to the large number of Californians suffering from often intractable polygenic diseases such as Alzheimer’s disease, autism spectrum disorders, autoimmune and cardiovascular diseases, diabetes, and respiratory disease. iPSCs can be generated from numerous adult tissues, including blood or skin, in 4–5 weeks and then differentiated to almost any desired terminal cell type. When iPSCs are derived from a disease-carrying donor, the iPSC-derived differentiated cells may show the same disease phenotype as the donor. In these cases, the cells will be useful for understanding disease biology and for screening drug candidates, and California researchers will benefit from access to a large, genetically diverse iPSC bank. The goal of this project is to reprogram 3,000 tissue samples from patients who have been diagnosed with various complex diseases and from healthy controls. These tissue samples will be used to generate fully characterized, high-quality iPSC lines that will be banked and made readily available to researchers for basic and clinical research. These efforts will ultimately lead to better medicines and/or cellular therapies to treat afflicted Californians. As iPSC research progresses to commercial development and clinical applications, more and more California patients will benefit and a substantial number of new jobs will be created in the state.
The applicant proposes to generate 9000 human induced pluripotent stem cell (hiPSC) lines from 3000 individual donors, using a combination of automated and manual processing steps. This derivation proposal was submitted as a collaborative effort with RFA 12-04 hPSC Repository Awards proposal IR1-06600. The for-profit applicant institution is based outside California, and the applicant proposes to set up a new hiPSC derivation facility in California. The applicant intends to use peripheral blood, and, if necessary, skin biopsies as a source of primary cells for derivation of hiPSC. The genes encoding reprogramming factors will be delivered using a genome integration-free method. The characterization of derived hiPSC lines involves gene expression analysis at the pluripotent stage, and verification of chromosomal integrity and of identity between the donors and their hiPSC lines will be accomplished by molecular analysis. Loss of the reprogramming vectors from the cells will be confirmed by polymerase chain reaction (PCR). For cell line tracking, the applicant proposes to use the laboratory information management system (LIMS) already in place at the collaborative RFA 12-04 applicant institution. Protocols - The overall plan from tissue collection through hiPSC derivation and characterization is very well described and thorough and provides confidence that the proposed effort can be achieved. - The proposed level of automation is appropriate, and the automation process has been well developed. - Reviewers appreciated the focus on peripheral blood as the cell source, as it will be generally easier to obtain blood than skin from tissue donors, especially children. The proposed expansion protocol for hematopoietic progenitor cells was also considered important. - The proposed molecular method to verify genomic integrity of hiPSC represents a valid alternative to classic karyotyping and is suitable for large-scale efforts such as this. - The proposal did not include a clear description of sterility and mycoplasma testing. - Newly derived hiPSC will be expanded to passage 5; reviewers were concerned that at this early stage the quality of the lines cannot be adequately determined. - While hiPSC characterization includes a pluripotency analysis, no test for differentiation capacity is proposed. Information from an embryoid body assay would give the end users some confidence that the cells have the capacity to perform in their experiments. Documentation and Quality Control - An excellent quality control system is in place at the applicant institution that will be transferred to the California location. It includes compliance and failure or problem resolution procedures which should add consistency overall. For instance, the applicant has considered potential issues that may cause reprogramming failure and has appropriate trouble-shooting steps in place to deal with such failures if they occur. - The applicant lists a complete set of standard operating procedures, which are already in routine use at the applicant institution. - The proposed use of the collaborator’s existing LIMS for cell tracking provides confidence in the accuracy of cell storage and retrieval and represents an advantage of near-immediate implementation and little personnel involvement at the California facility. Feasibility and Resources - The applicant institution has previously successfully generated and characterized large numbers of hiPSC lines using viral reprogramming as well as the proposed genome integration-free method. - The applicant institution is well resourced and has previously raised substantial funding. In support of their ability to deliver on this project, the applicant institution has already two large grants in a relevant area. However, these efforts are of a much smaller scale than the one proposed. A letter from one of the main customers as to their satisfaction with delivery or quality of the hiPSC would have been helpful. - A support letter from the institution that will provide the space and the space plans suggest a reasonable commitment and ability to set up a California location. For the size of this project, the cell culture space seems small, possibly warranting some reconfiguration of the space or inclusion of additional space. - The access to personnel and assets at the current location outside California during the start-up phase in California is a positive attribute of the proposal. The amount of experience in the field brought to bear on initial training and process development in California should facilitate tech transfer and efficient start-up. Project Director (PD) and Team - The PD has an impressive record of managing large cell culture-based scientific projects at the applicant institution and previously at other organizations. Of particular relevance to this program is the PD’s expertise in implementing automated cell culture protocols. - The breadth of expertise of the key personnel to be involved in this project ensures seamless implementation of the new facility and delivery on this commercial scale project. - The collaborations are a strength, especially the contemplated interaction with the IR1-06600 applicant for hiPSC banking. - Commitment by this out-of-state applicant to this project is clearly evident with the leasing of space, their investment in infrastructure and their plan to allocate individuals full time to this effort. - The proposed relocation of the PD to the California site will be essential to the efficient transfer of technologies and the start-up of personnel and facility activities. Budget - The budget items are well described and justified for the personnel involved, the scope of the tasks, and the subcontractor involvement; reviewers appreciated that a failure rate has been factored into the budget. - The proposed high investment in robotics is essential to be able to deliver the large numbers of hiPSC lines. - Reviewers expressed concern that some of the protocol design, e.g. culturing newly derived hiPSC to only passage 5, was proposed to lower cost. This is likely to lessen the quality of the end product, and CIRM should carefully evaluate in post award discussions, if funded, whether this is the best way to proceed.
- Derek J Hei