We propose to create a world-class cell manufacturing facility in California with an initial task of taking 3,000 donor tissue samples and creating induced pluripotent stem cell (iPSC) lines from the samples. iPSCs are extremely valuable as they are able to self-renew and can be converted into virtually all human tissues. In addition, since they are made from adult cells obtained from a specific donor, they can be used to model the biological profile of that donor, which is especially relevant if the donor has a particular disease. Therefore, iPSCs can be used to create cells or tissues that mimic disease and can be used in drug discovery efforts. To help make these efforts more efficient, we have developed a system that greatly improves the efficiency of making standardized iPSC lines. Typically, researchers generate iPSCs by hand, which limits the cells utility due to researcher variability and an inability to generate large numbers of cells. We seek to circumvent these problems through our system, which is completely automated from receipt of the tissue sample to eventual banking of large stocks of well-defined iPSC lines. We are able to generate very large numbers of cells from many donors, which we believe will facilitate the use of iPSC technology to discover treatments and cures for many diseases. The automated system we have developed is capable of generating all of the cell lines requested by CIRM, as well as many more for other investigators in California.
Statement of Benefit to California:
As a result of CIRM’s creation, California has established itself as a key player in all areas of stem cell research. If awarded this grant, our group will be able to integrate our unique high-throughput induced pluripotent stem cell (iPSC) derivation, characterization, and expansion platform into this rich environment. Based on our previous experience, our efforts to build innovative programs to change the landscape of stem cell research have enabled us to accelerate research and coordinate the stem cell communities at the institutions near our home location, which we hope to replicate in California. The use of iPSCs as a technology for basic biological research, drug discovery, and therapeutic applications has enormous potential, but requires major advances before translation. Typically, researchers generate iPSCs by hand, which limits the cells utility due to researcher variability and an inability to generate large numbers of cells. We seek to address these problems through our platform, which is completely automated from receipt of the tissue sample to eventual banking of large stocks of well-defined iPSC lines. We hope that our proposed program would coordinate activities between our home community and California to build a larger cohort of collaborators. These activities should allow California and its citizens to be among the first communities to see the innovations resulting from this research in the form of medical breakthroughs and commercial opportunities.
This application was provisionally recommended by the Grants Working Group. The applicant proposes to generate 9000 human induced pluripotent stem cell (hiPSC) lines from 3000 individual donors, using a fully automated process. The applicant also submitted a proposal under RFA 12-04 hiPSC Repository (IR1-06595) that is intended to complement this derivation proposal. The non-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 skin biopsies (preferred) or peripheral blood as a source of primary cells for derivation of hiPSC. A quarantine process is included to detect and exclude samples with mycoplasma or microbial contamination. The genes encoding reprogramming factors will be delivered using a genome integration-free method, and an alternate method, also genome integration-free, is proposed. The characterization of derived hiPSC lines involves gene expression analysis, both at the pluripotent stage and following differentiation into embryoid bodies, and verification of chromosomal integrity and of identity between the donors and their hiPSC lines will be accomplished by molecular analysis. For cell line tracking, the applicant proposes to use the laboratory information management system (LIMS) already in place at the applicant institution. Protocols - The proposed automation is, in principle, a strong advantage of this application as it enables high throughput production of hiPSC necessary for the scope of this project. - The proposed processes are well considered and the hiPSC characterization methods are appropriately amenable to automation. Proposed approaches are cost effective. - The applicant provides clear protocols for harvesting and shipping both skin punch biopsies and whole blood. Samples must be shipped fresh but this should not pose a major issue for samples collected within the state. - Reviewers appreciated that tests for microbial and mycoplasma contamination were proposed, and felt that doing so just during the initial quarantine phase for the source cells is a reasonable approach given the use of a feeder-free culture method and a high level of automation. However, reviewers suggested to perform sterility testing on the final cells for the first hiPSC lines produced to demonstrate that the system maintains aseptic conditions; and later on, periodic checks would also seem necessary to detect possible sterility failures. - There was some concern that the proposed hiPSC clone selection process could impact the quality of the resulting hiPSC lines. Preliminary data comparing hiPSC derived using the proposed clone selection process with hiPSC derived using a standard process would have alleviated this concern. - The applicant did not provide a detailed enough cell passaging protocol for reviewers to assess its potential effect on cell quality, and to determine whether the hiPSC will have been passaged enough prior to characterization to allow verification of full reprogramming to pluripotency. - The hiPSC characterization process is well thought out and is based on quick and efficient methodologies. The proposed assays have the capacity to definitively demonstrate the pluripotency status of the lines generated and the inclusion of freeze thaw viability is a plus. Documentation and Quality Control - The documentation process is well laid out; the LIMS system that is currently in use by the applicant institution provides complete documentation and represents a strong aspect of this application. - The quality control plan is reasonable. Feasibility and Resources - Although automation represents the future of large-scale hiPSC generation and its use is a strength of the application, it is not yet routine. Therefore, more detailed information on the proposed automation system is necessary to assess the feasibility of this approach. - The applicant provides supporting data demonstrating the capabilities of the proposed automated platform on a reasonable scale with a reasonable failure rate. However, evidence of the ability to perform large-scale derivations is still lacking. Letters from users documenting satisfactory delivery of hiPSC lines from this applicant institution would have partially alleviated this concern. - The 6-month proposed timeline for obtaining the new facility and for ordering, installing, and qualifying the requested equipment while hiring new staff is aggressive. It seems likely that time needed for completion of facility set-up will delay this program. - Reviewers were concerned whether the facility to be leased provides adequate space, both in regard to suitability for cell work and to overall size, especially if this applicant was also awarded the Repository Award. - The proposed hiPSC derivation platform would benefit from being established in one of the key CIRM-funded institutes; this would enable technical exchange and sharing expertise. Several world-class institutes are operating in California and they should be able to accommodate this project. Project Director (PD) and Team - The PD has experience establishing a hiPSC generation program similar to the one proposed and has, no doubt, the necessary skills to develop a successful project. - The commitment of the PD and the team structure are reasonable. - The scientific leadership of the applicant institution is world-renowned, with expertise in stem cell applications and in automation, liquid handling and quality control of cells. - Reviewers expressed concern about the fact that all new staff will have to be hired for the new facility. Resources from the existing team can likely be leveraged in helping to establish the California facility and team and it would therefore be useful for some personnel from the current facility to relocate to California for a limited period of time to facilitate set-up and initiation of the project. The applicant confirmed that trained personnel from the applicant organization will be present in California to train new staff until they are self-sufficient. - Evidence is provided that the applicant has worked to establish collaborations with a number of the top California hiPSC research groups. Budget - The applicant has laid out a reasonable plan to generate hiPSC lines of the requisite number at a cost that is justifiable. The salaries and the number of people listed are reasonable. - Almost one third of the budget is spent in the first year to purchase equipment. A more detailed description of this equipment would be needed to be able to assess the reasonableness of this budget item. - The total derivation cost per line, apart from equipment and indirect costs, seems reasonable, although some reviewers expressed concern that due to the large equipment budget, not enough funds would be available to derive 9000 hiPSC lines.