Stem cell biology and regenerative medicine holds tremendous promise for addressing critical health needs through early diagnosis of disease, identification of potential pharmaceutical agents, and generation of novel stem cell-based therapies. In this project, we seek to overcome one impediment to current efforts in stem cell biology and regenerative medicine and that is the shortage of well-defined, large collections of induced pluripotent stem cell (iPSC) lines that have been derived under standard conditions in the same facility with the same protocols. To accomplish this large task, we have assembled a team that has experience in deriving nearly 500 lines that include those from unaffected individuals, and those affected by cardiac anomalies, neurodegenerative disease, autism, schizophrenia, peripheral vascular disease, diabetes, premature ovarian failure (autoimmune and unexplained), and diverse and less common pathologies. We have chosen a method to derive the lines based on its reliability, efficiency, and cost-effectiveness. We also propose a well-defined set of measures to insure the high-quality of the lines and present a plan for success within the 3 year timeframe.
Statement of Benefit to California:
Stem cell biology and regenerative medicine holds tremendous promise for addressing critical health needs through early diagnosis of disease, identification of potential pharmaceutical agents, and generation of novel stem cell-based therapies. In this project, we seek to overcome one impediment to current efforts in stem cell biology and regenerative medicine and that is the shortage of well-defined, large collections of induced pluripotent stem cell (iPSC) lines that have been derived under standard conditions in the same facility with the same protocols. This project will enable unprecedented studies aimed at understanding human disease and tackling disease at its core. We focus our efforts on common conditions that affect many families in California including cardiac anomalies, neurodegenerative disease, autism, schizophrenia, peripheral vascular disease, diabetes, and also less common pathologies. The benefit to citizens will ultimately be realized through informed and earlier diagnosis combined with better treatments or more appropriate treatments based on knowledge of the disease/condition. Overall, the proposed research will contribute to our scientific infrastructure and technological growth, keeping California at the forefront, while contributing to advancement of better health care in individuals with diverse conditions.
The applicant proposes to generate 9000 human induced pluripotent stem cell (hiPSC) lines from 3000 individual donors using conventional, manual methods for cell handling and characterization. The non-profit applicant institution is located in California. The preferred strategy will utilize skin biopsies as a source of primary cells for hiPSC derivation, with peripheral blood-derived cells as a back up source. The skin fibroblasts or blood-derived cells will be isolated and expanded prior to reprogramming, and will be tested for mycoplasma contamination. Pluripotency will be induced by using a non-integrating vector to provide reprogramming factors. 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 will be accomplished by molecular analysis. Newly derived lines will also be tested for mycoplasma contamination. For cell line tracking, the applicant proposes to use an in house database. Protocols - The proposed approach for reprogramming somatic cells is rational and well justified, although some reviewers were concerned that the specific protocols used would prove less effective with peripheral blood-derived cells. - Reviewers strongly supported the use of skin fibroblasts as a primary cell source but recommended that a smaller biopsy be considered to avoid need for suturing, which could adversely affect patient consent. - The characterization approaches to be used are state of the art but provide a limited amount of information. Thus, reviewers suggested that a more judicious selection of differentiation markers could prove more informative. Documentation and Quality Control - Reviewers were concerned that the proposed modifications to the applicant’s existing data tracking system might not prove sufficient to serve as a true laboratory information management system (LIMS), which is critical for managing records and inventory of the thousands of cell lines and samples to be generated. - The proposal lacked information as to whether the proposed mycoplasma testing method is comparable to other approaches for detecting a complete range of species. - Reviewers noted that the proposed quality control measures, while adequate, include certain selection criteria that are extremely strict. Given normal variability within these parameters, it may prove challenging to systematically generate hiPSC lines that meet the proposed standards. Feasibility and Resources - From a technical standpoint, the application is highly feasible. The team has technical expertise, experience deriving hiPSC lines, and dedicated new space for this project. However, as the dedicated space would not be immediately available, the team would begin derivation in an existing space and subsequently move. The reviewers were concerned that the move may lead to delays in the project. - Without introducing some automation, at least for passaging and media changes, it is highly unlikely that the goal of generating 9000 hiPSC lines will be met. - Reviewers viewed the institutional support for this proposal as a significant asset. Program Director (PD) and Team - Reviewers were extremely impressed by the applicant team and considered them the major strength of this proposal. The PD has the experience to ensure success in the execution and administrative requirements for the proposed work, and the proximity of the team members to one another gives additional strength. - A reviewer noted that it would be critical to have a significant number of well-trained technicians on staff to conduct the labor-intensive processing steps in the proposed derivation pipeline. The reviewer expressed concern that the applicants might have underestimated the number of staff that will be required to generate 9000 lines, and that those proposed have not yet been hired. Budget - The application provides strong rationale for the various components of the budget, including staff costs. - Reviewers noted that the existing platform is already used in a broad number of projects and therefore tight management of resources and prioritization of projects will be required.